| Spoiler warning: The following contains plot details about|
the entire movie.
Pee-Wee enters Warner Bros. Studios in Burbank, California, disguises himself as a nun, and steals his bike back. In a wild chase scene, he flees from the Warner Bros. security staff through a variety of sets, causing havoc throughout the lot. Various actors and props, including a boat-shaped car, a Santa Claus sleigh, and a man in a Godzilla (Cleve Hall) costume, get swept into the chase. He also interrupts the shooting of a Twisted Sister music video for "Burn in Hell" from Stay Hungry. Using the gadgets on his bike when driving through the set of Tarzan and a western set, Pee-Wee manages to evade and fake out the guards and escape the studio. As he blissfully rides away however, Pee-Wee discovers a burning pet shop. After heroically saving all the animals, Pee-Wee faints on the sidewalk near the store's doorstep upon having to carry out the pet snakes just as the fire department and police department arrive. Though the fire department considers Pee-Wee to be a hero, the police decide to arrest him.
Pee-Wee is brought before a Warner Bros. studio executive who offers to buy the rights to Pee-Wee's story in exchange for dropping all charges and giving up their claim to his bike. Dottie is summoned into the office, bringing along Pee-Wee's beloved bicycle. He attends the premiere at his local drive-in theater, and it turns out to be a James Bond-style action film involving James Brolin and Morgan Fairchild fighting ninjas. Pee-Wee has a cameo appearance as a bellhop, with his voice dubbed over. After watching for a few minutes and outsmarting Francis, Pee-Wee decides to leave, having already lived the real story. Reunited with his bike, he rides away with Dottie, happily ever after.
|All spoilers have been stated and have ended here.|
- Paul Reubens as Pee-wee Herman (billed "Pee-wee Herman as himself"): The titular protagonist. A strange man who acts like a child. He sports a gray flannel suit with a red bow tie and clean-cut hair. He is very obsessive over his bicycle, traveling across America in search of it.
- Elizabeth Daily as Dottie: The deuteragonist. She helps run a bike shop and also has feelings for Pee-wee, though Pee-wee declines to go out with her.
- Mark Holton as Francis Buxton: The main antagonist. A fellow man-child like Pee-wee, his neighbor, and rival. He is very spoiled. He offers to buy Pee-wee's bike, but Pee-wee refuses. Francis hires someone to steal the bike before it is purchased by Warner Bros.
- Diane Salinger as Simone: A waitress that Pee-wee meets in Texas. She develops a friendship with Pee-wee and yearns to live in France. Her violent boyfriend Andy flunked French in high school, and therefore dislikes France. At the end of the film, Simone is dating a French man named Pierre.
- Judd Omen as Mickey Morelli: A fugitive Pee-wee meets on his way to Texas. Mickey is an escaped convict on the run from the law because he cut off a "do not remove under the penalty of law" mattress tag. He also has a bad temper and abandons Pee-wee for his safety. However, Pee-wee ends up inviting Mickey to his movie.