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| Paul Reubens | Pee-wee Herman |
| Elizabeth Daily | Dottie |
| Mark Holton | Francis |
| Diane Salinger | Simone |
| Tony Bill | Terry Hawthorne |
| Cassandra Peterson | Biker Mama |
| James Brolin | Himself - as PeeWee |
| Morgan Fairchild | Herself - as 'Dottie' |
| Jan Hooks | Tina |
| Judd Omen | Mickey |
| Monty Landis | Mario |
| Daryl Roach | Chuck |
| Starletta DuPois | Sgt. Hunter |
| Professor Toru Tanaka | Butler |
| Ed Herlihy | Mr. Buxton |
| Ralph Seymour | Francis' Accomplice |
| Lou Cutell | Amazing Larry |
| Erica Yohn | Madam Ruby |
| Jason Hervey | Kevin Morton |
| John Paragon | Movie Lot Actor |
Childlike Pee-wee loses his vintage bicycle and embarks on a cross-country adventure to get it back.
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