When Pee-wee leaves the park and is driving his bike down the road, there is a red classic Mustang on the right side. In the next frame, we see Pee-wee coming down the same road from the same angle, but there is now a blue car where the Mustang was.
When Pee-wee and Francis emerge from the battle in the swimming pool, Pee-wee's face is a little wet but his clothes are completely dry.
The Criterion theater, in the mall that Pee-wee goes to, advertises that they will be showing something called "Cartoon Cavalcade." But a few seconds later, in a reflection, the theater sign is promoting an entirely different feature. When he starts walking off not long after this the Cartoon Cavalcade signage is back.
In the bicycle chase scene where a studio guard pursues Pee-wee, the guard pulls one of the tassel handles off Pee-wee's bike. In the close-up shot, Pee-wee presses a button that causes a handle without a tassel to spring out. When they cut back to the wide shot, the tassel is back as well.
When the 1959 Edsel convertible goes over the cliff, the rear quarter windows go from being down, to up, and then down again.
When Pee-wee and Simone watch the sunrise they enter a red T-Rex, but exit a green T-Rex, because it was being lit with a red spotlight.
When Pee-wee goes to "Mario's magic store", the glass door shows the "Mario's" part of the sign on the glass facing into the shop, while the "magic shop" part of the sign is shown correctly facing outside. This was likely intentional. After all, it IS a magic shop.
While making breakfast, when Abraham Lincoln flips the fifth and final pancake which sticks to the ceiling, you can see the pancake hit the ceiling and bounce to the side, revealing that the filmmakers simply dropped pancakes on the floor then inverted the shot.
When Pee-wee enters the magic shop, the image is flipped (most likely to facilitate being able to read "Mario's Magic Shop" on the shop door), as a result, the hanky in his left-hand suit pocket is switched to the right.
When Pee-wee's breakfast is being made, the orange being squeezed in the juicer is clearly a foam-rubber ball.
When Pee-wee removes his bike chain from its container, the chain is clearly seen begin fed through a hole in the bottom. This is only visible in unmatted full-screen home video.
Matte lines visible around Pee-wee when he does his bike jump.
At the beginning of the chase scene on the studio lot, the security carts are unplugged from the wall as if they were electric carts, but then they are heard making noises as though they were gas-powered.
When Pee-wee is driving Mickey's car past the road warning signs, the signs are clearly seen attached to carts on rollers coming towards the camera. This is only visible in unmatted full-screen home video.
When Pee-wee goes to get his bicycle from its hiding place at the beginning of the movie, we see gray trousers of a crew member, and someone's hand inside the left side of the door.
During the rodeo scene, the back end of the mechanical bull Pee-wee is riding is briefly visible.
A sound guy is visible in the window at the concession stand when Andy chases Pee-wee from the bus station to the parade.
Near the end of Pee-Wee's chase scene at the Warner Bros. studios, he activates a launch mechanism on his bike and soars over a barb wire fence onto a roof; a crew member wearing sunglasses can be briefly seen on the lower right hand side of the screen.
Snow-capped mountains in the background in various Texas scenes.
Pee-wee calls from a Pacific Bell payphone in a Texas biker bar.
The exact same buildings are visible in the background when Pee-wee and Hobo Jack begin singing on the train, and (supposedly) much later when Pee-wee jumps off the train.
When Pee-wee and Mickey are saying "the law" back and forth, the scenery to the right of Pee-wee changes abruptly from a lush meadow with trees to a huge rock wall.
The date at the truck stop is established as being January 11, yet only a few days later a Day of the Dead (November 1) parade is seen taking place while Pee-wee is in San Antonio.
The car that Mickey was driving was either registered to him, or even more likely stolen. In either case, any police inspection would reveal this, making their disguises irrelevant.
Pee-wee accidentally hits his head on the ceiling during the Tequila dance scene.
When Twisted Sister are filming their music video, someone is following them with a boom mic, which is unnecessary as the band is using the pre-recorded album version of the song for the music video.
When Pee-wee knocks the motorcycles over, one of them has no cap on the gas tank.
Bulls are actually attracted to movement, not red.
Dottie calls Peewee "Peepee" at one point.