Jonathan slowly coming to the realization that he and his kindly old aunts have murdered the same number of people.
Especially the ensuing "score tallying" scene:
Dr. Einstein: "Johnny, don't brag! You've got twelve, they've got twelve; the old ladies are just as good as you!"
Meanwhile the aunts are looking quite flattered.
And of course, his frustration that while he's been hunted like a dog for years due to his murders, his aunts have never been caught and are still living their happy lives.
The aunts' latest "charity case" is handed a glass of the elderberry wine while Mortimer tries to reach the sanitarium on the phone. After hanging up, he distractedly grabs a glass of his own. The aunts warn him off, he realizes what he's doing and puts the glass down...and seeing the visitor about to drink, lets out a piercing shriek of horror.
"Insanity runs in my family. (beat) It practically gallops!"
Dr. Witherspoon's reaction to it is just as funny:
Lt. Rooney: Look here, doc, couldn't you also make room for the ladies (at Happy Dale with Teddy)?
Dr. Witherspoon: *Just* the ladies?
Mortimer's increasingly funny Sanity Slippage. He goes from suave, assured on-top-of-the-world Only Sane Man, then discovers he's the Only Sane Man (because for that to happen, there's the rather unpleasant discovery that everyone else is insane, including all of his family, and it may be hereditary), and the rest of the movie is basically him not dealing well with that information while managing about three or four plotlines at once. By the end, he's been morphed into a high-strung, Seen It All man who's over the moon to find out he's not in hereditary danger of a mental disease, but may have been driven to one anyway.
Teddy's initial reaction to meeting Dr. Witherspoon:
Teddy: "Is he trying to move into the White House before I've moved out?"
Mortimer: "Who, Mr. President?"
Teddy: (points accusingly at Witherspoon) "Taft!" (Note to directors: Be sure to cast a fat man as Dr. W.!)
After being confronted by her nephew Mortimer about the twelve men she and her sister have murdered, Aunt Abby huffily informs Mortimer that she "would never stoop so low as telling a fib!"