At the beginning of each show, viewers and the Playhouse denizens were told the day's "Secret Word" and were instructed to "scream real loud" every time a character on the show said the word, which was given to Pee-wee by Conky, his trusted and beloved robot. In the first season, the Secret Word was printed on a sheet of paper and capitalized when flashing on the screen. From the second season onward, the word was printed on a card. In the second and third seasons, the word flashed in all lowercase letters on the screen. In the fourth and fifth seasons, the word flashed in all capital letters. Immediately after receiving the Secret Word, Pee-wee would try to demonstrate the usage of the word by usually trying to trick someone in the playhouse into saying it, usually followed by Pee-wee accidentally saying the word himself. The word was always used continuously throughout the episode, and Pee-wee always said it right before he got onto his scooter at the end of the show. Mrs. Steve hated the Secret Word routine, as she hated anyone or anything childish; Pee-wee himself also objected to the Secret Word routine in four particular episodes, the first three of which he harshly demanded silence from his friends, and the last was when he asked everyone what the screaming was for in the final episode after looking at them funny. Additionally, after everyone screams to the Secret Word if Pee-wee says it, on some occasions he will shake his head in frustration and disbelief, including him rolling his eyes (as if the routine is getting on his nerves).
On several occasions, someone other than Pee-wee would perform this routine because Pee-wee was unable to do so himself, such as Miss Yvonne in the final episode and Magic Screen in Pajama Party. In Sick? Did Somebody Say Sick?, Randy menacingly instructed everyone to "bark like a dog", rather than "scream real loud" because he made a rule against screaming when he played "Randy's Playhouse," and this made Pee-wee extremely angry but presumably relieved any parents of young viewers.
- One of the criticisms that many parents have with Pee-Wee's Playhouse is that children would keep screaming anytime a character of the show said a secret word for the entire week, until another episode aired and another secret word was unveiled. Parents and older children would get irritated and disturbed, like Pee-wee did in Pee Wee Catches a Cold or Love that Story and Mrs. Steve did in Ice Cream Soup, at children screaming because they would want absolute quiet from them.
- This routine would be referenced in the 1996 Power Rangers series Power Rangers Zeo (in the episode "The Puppet Blaster") with Captain Pete and his friend Puppetman.
- The idea of using a "secret word" actually was created by world-famous comedian Groucho Marx for his show You Bet Your Life. The response, however, was different.
- Some young and old viewers thought the secret word is called the "word of the day". Paul Reubens took advantage of this in a commercial when it was going to be shown on Canadian television, and some people still call the Secret Word the "word of the day".