Whenever it is pitch dark, characters' open eyes are always visible.
The same applies, eventually, to any great big teeth which something lurking in the darkness may possess. A common shot is to have a nervous character scared in the dark as a variety of seemingly glowing teeth gather around them.
In live action, it is usually used to spooky effect.
By the Lights of Their Eyes seems like it should always go with Glowing Eyes, but it really doesn't.
This is generally a form of Hollywood Darkness, and may immediately precede a Nuclear Candle as part of The Reveal.
Depending on how it is drawn, can be Lazy Artist. While drawing the scene in the page image takes as long as normal, when a cartoon simply shows the character's eyes in pitch black it saves a lot of animation time.
A Sister Trope of The Darkness Gazes Back. Not to be confused with See the Whites of Their Eyes, which is a figurative expression for getting close to your enemy. Nor Eye Lights Out, which is a death trope.
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Anime & Manga
Kinnikuman: In Episode 2, when the eponymous character trips on a car while giant and flies face-first into a subway entrance.
Snarf Quest in Dragon magazine #105. When Telerie, Snarf and Aveeare are exploring a cave in the dark, you can see their eyes in the darkness. Then another pair of eyes appears and a growling noise comes from them. Snarf starts stabbing at them, and when Telerie lights a torch they see that he has killed a monster.
The monsters under Calvin's bed usually are only heard and not seen, but in a few strips their eyes were visible this way.
Hobbes occasionally has his eyes glow in the dark when hunting Calvin, crossing this over with Glowing Eyes of Doom. (Vaguely justified in Hobbes's case, as feline eyes actually have a layer of shiny reflective cells to help them see in the dark — see Real Life below.)
An entire week of Garfield strips took place during a blackout once, with only Jon's and Garfield's eyes visible.
Films — Animation
Although common enough in Disney short cartoons, this trope is rarely used in the feature films, perhaps because they generally try for a more realistic tone. A few exceptions:
Treasure Planet, when B.E.N. accidentally cuts off the power on the ship. (Justified in that his eyes are actually light displays.)
Alice in Wonderland (1951). When Alice is looking at the Tulgey Wood sign, a number of eyes appear in the dark above it. One pair is revealed to be a "glasses bird", which hops onto Alice's head.
Snow White and the Seven Dwarfs has a rather scary example. When the title princess runs in panic in the forest, some yellow glowing eyes seems to stare at her. They actually belong to friendly cute animals but Snow White's imagination makes them look like evil and demonic.
In the CGI feature Robots the characters have actual headlights for eyes — possibly a Reconstruction, although it may not have been intentional.
The Thief and the Cobbler: The end scene where Zig-Zag is eaten by crocodiles is a wonderful example of playing with this trope, especially the final scene, where Zig-Zag's eyes are first encircled then blacked-out by white teeth.
In An American Tail: Fievel Goes West, Fievel is chased into a hole by a hawk, and we only see his eyes... accompanied by evil glowing eyes that belong to a scorpion.
The Catbus from My Neighbor Totoro, whose eyes serve as headlights, and has small mice on its roof and lower body whose eyes serve as the destination sign backlights and taillights, respectively.
Justified in WALL•E, as the robots' eyes are actual lights.
When in the sewers in the Doom movie, Goat approached a set of eyes in a dark corner after his flashlight dies, while looking for a scientist. When he asks "Doctor Willets?" several other eyes appear around the main set, revealing not that there are more of them, but that the transformed Dr. Willets has grown several more eyes all over his head. The creature then leaps and attack Goat.
Also done in Pitch Black, where it was a character point.
When Pee-Wee Herman ends up abandoned in the desert in Pee-wee's Big Adventure, his flashlight goes out, leaving only his eyes... then several other eyes. When he gets his flashlight back on, he's not met with the happiest sight.
In 300, the emissary of Xerxes bribes the priests of the oracle, then fades to a silhouette with only his eyes remaining.
Emperor Palpatine from Star Wars usually keeps his hood pulled forward enough to cast a dark shadow on the upper half of his face, but occasionally the light glints off his eyes creating a very spooky effect.
In For a Few Dollars More, when Manco is creeping around the room where El Indio hid the cabinet, this effect is achieved by painting Eastwood's face dark brown, so his eyes glow out.
The second Astérix movie has a sequence with the main characters stuck in one of the Pyramids of Gizah. When their torches go out, they suddenly turn into cartoon eyes floating in the dark (and stay that way until the dog leads them out).
In the 1974 Black Christmas, the "Prowler" is never truly seen out of shadow. The only scene in which you view him, is where he is peering from behind a door. Ominously, all you see is an eye, the rest hidden in shadow.
In Scary Godmother, Jimmy and his friends hide in a closet after being scared inside the haunted house.
In Oz: The Great and Powerful, Oscar, Finley and China Girl are spied on by a whole lot of pairs of gleaming eyes when they enter the Dark Forest. Several times they turn around suspiciously, but are an instant too late to spot the eyes until the snappy animated plants they belong to are ready to attack.
Lone Wolf: An interesting variation occurs in The Kingdoms of Terror. Lone Wolf is being attacked by a very fast monster with glowing eyes in a pitch-black castle. If you choose to fire an arrow at its eyes you actually get a bonus to your success roll since, because they're glowing, they're an easier target.
The alien atevi of the Foreigner series have reflective retinas (just like real-life cats), causing their pupils to shine in dim light; this unnerves a lot of humans. Conversely, the fact that the eyes of humans don't shine in dim light unnerves a lot of atevi.
Done rather creepily in the (live-action) Dune miniseries: all the Fremen have glowing blue eyes. In the sequel miniseries the brightness is pulled back a little. (In the books, the eyes of spice addicts are so dark-blue they appear nearly black.)
Done rather creepily on The X-Files with Eugene Victor Tooms, the liver-eating mutant whose eyes glow a dull yellow.
The Goa'uld of Stargate SG-1 do Glowing Eyes of Doom. It is done in the dark on occasion, and once by a villain as he stepped into the dark, so his eyes are the last thing to go into the shadows.
A rather unsettling number of examples come from Chromeo's music video for "Don't Turn the Lights On," at the end culminating in the lights coming back on showing just the lead singer's floating eyes. Which, given the rest of the video, is a bit more humorous.
When night falls on Muppet Treasure Island, the characters' eyes glow. As do the eyes of the wild boar tribesmen sneaking up behind them...
Used in Sesame Street, when Maria has to go into Oscar's trash can, in order to hide what the actual interior looked like. Oscar's glowing eyes were identical to his normal ones, though Maria's were clearly Muppet approximations.
In one classic Muppet sketch, Ernie and Bert run so many appliances in their apartment that it blows a fuse, and the blackout is presented with their eyes floating around in complete darkness.
During one of the animated sequences in the show Fantasmic!, a late night performance at one of the Disney Theme Parks, the character Mickey, during a voiced version of the sequence from "The Sorcerer's Apprentice", goes down a whirlpool, after which you see his eyes appear in a dark place for a couple seconds as he tries to figure out where he is.
Journey into the Dark is an attraction from the Futuroscope that put you into the shoes of blind people. The only thing you'll ever see in the darkness is the glowing eyes of the Yeti at the end of the walkthrough.
One of the early BIONICLE animations showed this when a bunch of Onu-Matoran were trapped by a cave-in. Possibly a Justified Trope, since most incarnations show the characters' eyes having some sort of glow, even in daylight.
In King's Quest IV, Rosella at one point must navigate a pitch-black cave with a resident troll and a chasm opening into a Bottomless Pit ... all without any light for the player to see by.
In the large labyrinth of King's Quest VI, one room is so dark that all the player can see of Alexander is his white eyes against the black surroundings. If the player doesn't have a source of light, rumbling noises and a growl are heard as the Minotaur enters the room, while Alexander's eyes are lifted up, shaken, and... well, that's gotta be painful.
In Rayman 2, there are several instances in which there are inaccessible pitch-black rooms, holes in the walls and half-closed coffins where if you look into them, you're sure to see a pair of eyes looking back at you.
Rayman Origins and Rayman Legends also use this trope: The former in the introduction to the game, and the latter on the silhouetted loading screens.
This is utilized during the final battle of Ōkamiden in that one of the attacks of the Big Bad is to cloak the battlefield in darkness, and his glowing eyes are the only way to find him, attack him until he's stunned, and then bring the light back.
In S.T.A.L.K.E.R.: Shadow of Chernobyl, ALL of the mutant's eyes glow. No matter what. Even AFTER they're dead.
MetaKnight in Kirby. While his face is shadowed by his mask, he has shiny eyes.
Taokaka from BlazBlue has her face concealed in a hood, showing only nondescript red eyes and a crescent grin with dagger teeth.
In Tales of Monkey Island Chapter 4: The Trial and Execution of Guybrush Threepwood, one of the buttons on De Singe's Auto-Trepanation Helmet causes the entire "laboratorium" to go dark temporarily, with only Guybrush's eyes visible when he says, "Ack! I'm blind!" Thankfully the lights turn back on.
Stepping into an unlit room in the caves will cause Brandon's death by a horde of glowy-eyed monsters.
Parodied in Malcolm's Revenge. Leaving Darm's Hut at nighttime will cause red eyes to appear behind Malcolm... and step out of the shadow to reveal squat imp-like creatures holding signboards depicting glowing red eyes. They smack Malcolm in the head with a signboard.
Zork is infamous for Grues — creatures that will kill you in the darkness, yet you never ever see or hear a description of what they look like as they seem to only exist within darkness. In Zork Zero, though the game is still all text like previous games, you get snippets of images now and then. Such as you can play a card game with The Jester, one of the cards is "Grue" which shows nothing more than a pair of eyes in the darkness).
Dark Souls has this in the Tomb of the Giants, in which there is a supernatural darkness that severely limits your sight range. If you look, though, you can see the skeleton enemies' Glowing Eyelights of Undeath in the darkness. There is also Manus in the Artorias of the Abyss DLC, whose (many) eyes can be seen in the distant dark from outside the boss room if you look in just the right place.
At one point in the third trial of the third Phoenix Wright: Ace Attorney game, there is a power outage, and you can see Phoenix's and Godot's eyes (though, given the fact that Godot's "eyes" are an illuminated visor, it's probably justified). In fact, this is foreshadowing of the fifth case, in which Godot's illuminated visor becomes vital testimony for a murder which took place in pitch darkness. Phoenix's eyes, not so much.
Shows up on Homestar Runner in the Strong Bad Email your edge, providing both the former page quote and an unusually detailed look at Strong Bad and The Cheat's eyes.
Strong Bad: Whoa, where are we? I suddenly feel so... easy to animate.
In the Barbie: Life in the Dreamhouse episode "Closet Princess", when Teresa and Nikki slide through a dark passageway, they look like eyeballs floating against a dark backdrop.
Shows up in Breakpoint City. During a power outage, Sofia's eyes are visible as usual. Ben's glow blue, which he explains is because of lights in his glasses. Then Dan comes in, with regular eyes — and glowing green teeth.
Ben: For the love of God, Dan, brush your teeth.
Done and lampshaded in the Insecticomics. This causes confusion at once point, where Tarantulas mistakes the snake-handed Quickstrike for being two mechs because he sees two pairs of optics.
In Goblins, this is one of Thunt's favourite effects. When a character is seen in silhouette, you can generally see their eyes, mouths, significant scars, and occasionally magical effects. In general, their major identifying marks — you see Chief's tattoo, for instance, or Kore's helm's eye-hole.
A particularly notable example is the Bugs Bunny cartoon Hyde and Hare (1955), where Bugs locks himself in a dark closet with Dr. Jekyll to protect them from "the maniac". Jekyll, naturally, transforms into Hyde — and it's only seen by the animation of his eyes turning huge, red, and monstrous.
Spoofed in an episode of Tiny Toon Adventures, where Buster and Plucky get stuck in a dark pit, and pass the time by using their illuminated eyeballs to play Pong and Pac-Man. The best part of that scene was when Buster and Plucky lit a match, and when the room was lit, all that remained were their eyeballs.
Lampshaded in "A Fish Called Selma" when Homer asks, "Marge, could you close your eyes? I'm trying to sleep."
Spoofed in the final scene of "The Last Temptation of Homer". Homer and Marge are in a hotel room, and after he turns off the lamp for some happy time, a third set of eyes appears, those of the creepy bellman from earlier who starts cat-calling. Homer's eyes become angry and he punches the bellman, and one of his eyes disappears.
In the Treehouse of Horror XIII segment "The Island of Dr. Hibbert": Two pairs of eyes peer out from under some bushes. One pair turns into a set of fangs and devours the other pair.
In "Mother Simpson", after the power is cut from the house, Marge scolds Homer for faking his death, in pitch blackness. Her emotions and body language are wonderfully portrayed through only her eyes.
There's another episode where not only Homer's eyes are visible in the dark so are his teeth.
One Couch Gag showed the family's eyes rushing to the couch in darkness, and then the lights turn on, revealing that they're all just eyes, and then the (eyeless) family joins in, at which their eyes immediately place themselves onto their faces.
"A Test Before Trying" has one of the rare (or maybe not — see pic above) examples where the scene isn't completely pitch dark.
Lampshaded in "Diggs", when Marge tells Homer that the brightness of his eyes are keeping her up.
Classic Disney Shorts: Mickey Mouse himself in his pie-eyed days. While at face value his eyes seem to be the two black inkblots on his face, in the dark (and in the way these inkblots move around his head and in some colorations of the comics) it's revealed that they're actually two pupils in a merged set of giant eyeballs that take up half his face. His design from Plane Crazy without the eyes outlined. That, or maybe they just couldn't think of any other way to make those black eyes to show up in the dark.
Parodied in the Roger Rabbit cartoon Roller Coaster Rabbit, where at one point the eyes are running around on little legs.
Used in Metalocalypse when the members of Dethklok snuck into a closet to eat food, since they were supposed to be on diets.
Ruby Gloom has fun with the characters' distinctive eyes. They cheat a little with Misery's inverted eyes (white pupils, black sclera) by turning them gray.
The Herculoids episode "The Lost Dorgyte": the eyes of the monsters of the Stone Forest.
Parodied in Drawn Together, when Captain Hero and Unusually Flexible Girl have sex. The lights go off leaving just their eyes visible as they go through various positions. The positions start off reasonably enough, getting more and more unusual until their eyes are going through a Newton's Cradle type motion.
"The Power Pirate". Wendy and Marvin are trapped in a dark room, Initially all the audience can see is their eyes.
"The Planet Splitter". When Doctor LeBon's assistant closes the observatory's door and shuts off the outside light, we see Wendy's eyes floating in the darkness.
"The Shamon U". While Wendy, Marvin and Wonder Dog are hiking up Mystery Mountain in the dark they see two pairs of glowing eyes approaching them. The eyes turn out to belong to Batman and Robin, who were following them.
Used in an animatedSesame Street sketch in which two people work together to turn on a light in a dark room. When the light is turned on, the first person sees that the second person is a repulsive-looking creature, and turns the light back off.
In The Real Ghostbusters episode "Knock Knock", the Ghostbusters enter the lightless Place of Lost Souls. First we see the Ghostbusters' eyes...then we see the eyes of the dozens of beings surrounding them. We never do find out what any of them look like.
Done rather nicely on Teen Titans when the Titans fight a bunch of machines in a dark sewer-like area. Starfire's green energy bolt is the only source of light. Beast Boy, in human form, is cornered by a machine. When the light goes away, his pupiless eyes remain. When the light comes back, it's revealed he turned into a gorilla and crushed the machine.
The Fairly OddParents episode "Lights Out" is based around this trope, granted that the episode takes place in complete darkness for a good chunk of the episode, with no filters whatsoever.
Johnny Test used it twice, with the family hiding in the closet in "Johnny Zombie Tea Party" and in "Johnny Trick or Treat" when Johnny, Dukey and Jillian fall into a pitch-dark basement, with their eyes visible.
This can happen when using a webcam in a dark room, when the only light sufficient enough for the webcam to pick up is bounced off the person's eyes and teeth.
Nightvision makes people's eyes seem to glow in the dark in an otherwise murky green haze.
Many animals, especially deep sea and nocturnal animals (including felines and possums), have what is called the tapetum lucidum, a layer of tissue at the back of the eyeball that reflects light, improving the animal's vision in dim conditions. This can make the eye appear to glow if a light shines directly at it. The reflector "eyes" of a bike work and the "Cat's eye" for roads function about the same way.
Possibly inspired by this is that it used to be believed that light came from someone's eyes and that was how they saw but this an old (500 BCE old) theory.