- The entire climax of the film with Pee-Wee's rampage across the Warner Brothers studio lot. Moments include:
- Pee-Wee's argument with Francis.
Pee-Wee: I wouldn't sell my bike for all the money in the world. Not for a hundred, million, billion, trillion dollars!
Francis: Then you're crazy.
Pee-Wee: I know you are but what am I?
Francis: You're a nerd.
Pee-Wee: I know you are but what am I?
Francis: You're an idiot!
Francis: I know you are but what am I?
Together: I know you are but what am I? IknowyouarebutwhatamIIknowyouarebutwhatamIIknowyouarebutwhatamIIknowyouarebutwhatamI...
Francis: No, I'm not.
Together: You are! No way! Knock it off! Cut it out!
Francis: Oh shut up, Pee-Wee.
Pee-Wee: Why don't you make me?
Francis: Why don't you make me?
Pee-Wee: Because, I don't make monkeys, I just train them.
Francis: Pee-Wee, listen to reason. (Pee-Wee put his hand on his ear to "listen") Come on!
Pee-Wee: Shhhh, I'm listening to reason.
Pee-Wee: That's my name, don't wear it out.
- Pee-Wee's friend who owns a magic shop pulls out a big head with an Evil Laugh.
Pee-Wee: (backs away) NOOOO!!! Ha ha ha ha!
- The bath fight with Francis is hilarious.
Pee-Wee: (after locking the bathroom door) Go ahead; scream your head off! We're miles from where anyone can hear you! (maniacal laughter)
- Pee-Wee scares some local punks by making an animal sound.
Punk: Hey, man! You're new in this turf. We don't take kindly to strangers around here!
Pee-Wee: *HISSSSSS!* (the gang runs away)
Madam Ruby: Alright, you wanna wear a wet jacket, it's fine with Madam Ruby.
- Even funnier, the punk who gets in his face to threaten him is a Creator Cameo from Tim Burton!
- Still funny later on when Madam Ruby tries to remove his wet jacket and he hisses at her as well, causing her to recoil quickly.
- Pee-Wee throws his hitchhiking thumb away when it didn't work.
- The hobo keeps singing and singing.
- When Pee-Wee wanders the wasteland at night (with just his eyes showing), he breaks out one of his toys: Illuminating goggles. But when he puts them on...
Aaaaaaaaaaaaaaaaaah! (He's surrounded by obviously fake animals, and a few real ones.)
- Pee-wee's impatience in the Alamo tour.
- "I'M TRYING TO USE THE PHONE!"
"I barely touched them!"
- Oh my God, just that whole scene at the biker bar.
- Pee-Wee cries after he accidentally knocked the motorcycles.
- When Pee-Wee makes friends with the bikers and they let him borrow a bike. He gets on, revs the badass motor, rides off and... smashes right into a sign.
- While on the phone with Dottie, Pee-wee has to convince her that he's at the Alamo. He does so by loudly singing "The stars at night, are big and bright—" ... Cue the ENTIRE CROWD walking by on the sidewalk stopping, turning, clapping four times, and singing "Deep in the heaaaaaart of Texas!" in perfect unison.
Cowboy: (pulls Pee-Wee up) That's you, cousin!
- Also about Texas, after a rodeo, Pee-wee says he doesn't remember anything...but he remembers the Alamo. Everyone around him cheers.
- While hiding from Andy, Pee-Wee disguises himself as a cowboy. Even walking in a cowboy strut. He found out too late he was dressed as a bull rider.
Pee-Wee: Oh noooo.
- The chase through the soundstages and backlots, particularly as "bystanders" get swept up in it.
- "Is this something you can share with the rest of us, Amazing Larry?!"
- The funny look at Pee-Wee's face when he honks his horn at everyone. Followed by a suspiciously brief Death Glare similar to the T-1000's.
Pee-wee: Exhibit D: Jimmy what is this? Too late! Chip!Chip: It looks like a pen.Pee-wee: Exactly! I bought this pen exactly one hour before my bike was stolen. Why? What's the significance? I DON'T KNOW!
- "Exhibit Q: a scale-model of the entire mall! X marks the scene of the crime. These arrows here show the exact position of the sun at the hour of the crime. Jupiter was aligned with Pluto! The moon was in the seventh..."
- Pee-Wee finally acts against his snake phobia at the burning pet shop.
- The very first scene, as Pee-Wee pulls to the lead in the Tour De France on his coaster bike in his gray suit and tie - sets the mood for the whole movie.
- And when they finally pulled the driver's body.. from the twisted.. burning.. WRECK, it looked like this!, doubles as Nightmare Fuel as well.
- That look Mickey —who just got out of prison, mind you— gives to Pee Wee while he's disguised in drag is both creepy and hilarious at the same time.
- "Paging Mr. Herman..."
- Right after the car makes a soft landing:
- Before that, as they were falling down screaming, they both hold onto each other. Mickey then pushes him away while still screaming.
- Pee-wee trying to get into Francis' house.
Pee-wee: I wanna see Francis.
Butler: Francis is busy.
Pee-wee: Busy doing what?
Butler: Busy having his bath.
Pee-wee: Really, where are they hosing him down?
(The Butler slams the door, but Pee-wee knocks again and the Butler answers again)
Pee-wee: Me again.
(The Butler slams the door again, but hears another knocking sound. He opens the door, but finds a knocking device attached to it, while Pee-wee sneaks by behind him.)
- The butler looks like Oddjob and Francis' bathtub is basically an indoor swimming pool.
- The deadly serious reason why Pee-Wee keeps avoiding Dottie's attempts to ask him out to the drive-thru.
Pee-Wee: Déjà vu.
- And the Call-Back when the same speech is given by James Brolin. Extra funny points for turning "I know you are, but what am I?" into a pick-up line.
- And from Mickey.