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They appeared in The Adventures of Super Mario Bros. 3 once, in the episode "Kootie Pie Rocks". In the episode, Cheatsy overhears the Mario Brothers talking about going to a rock concert with Princess Toadstool, for Milli Vanilli. Cheatsy made his way back to Castle Koopa and explained to Kootie Pie Koopa what the Mario Brothers intended to do. Kootie Pie was not pleased about the Princess getting something that she wasn't. In the middle of the band's concert, Cheatsy and King Koopa appeared in their Doomship and kidnapped the two singers, then made their way back to Castle Koopa. Kootie Pie wanted the two rock singers to sing for her but they refused. Kootie Pie was enraged, and she turned the two into accountants. When the pair tried to escape, the Sledge Brothers foiled their escape, and Kootie Pie said that they would remain in Dark Land forever unless they do her personal concert. Milli Vanilli said they could not perform for Kootie Pie even if they wished; for their songs required a backup band. Kootie Pie then screamed to Bowser that he should have kidnapped the instrumentalists as well.
Mario, Luigi, the Princess, and Toad took advantage of this and disguised themselves as a band. Princess fooled Kootie Pie into giving back Milli Vanilli their true forms as she was questioning why "two accountants" would be singing. The quartet helped Milli Vanilli escape from the castle, then returned them to the Real World. Kootie Pie protested their escape, as the guards ran off in disgust and Bowser came in wearing earmuffs, saying she can scream all she want as that is preferable to that lecherous music he just heard. Kootie Pie is surprised by Bowser resisting her threats of a tantrum, and then lays down the law by confining her to her room, saying he has had enough of her temper tantrums.
Back in their proper concert in Brooklyn, Milli Vanilli dedicated their rescue to Princess Toadstool.
Milli Vanilli was the only guest appearance on The Adventures of Super Mario Bros. 3; whereas the preceding show, The Super Mario Bros. Super Show!, had several during the live-action segments.
Milli Vanilli was accused of lip-synching, and the duo admitted to this. Glitches in their equipment revealed this only two weeks after the initial airing of "Kootie Pie Rocks", and one week after the group won the 1990 Grammy Awards, which the duo relinquished. Subsequent airings of the cartoon cut their songs and replaced them with different instrumental music. (Curiously, the pair still moved their lips while performing these wordless songs in the re-releases.) One of the episode's references to a Milli Vanilli song was also removed, and Milli Vanilli's song dedicated to Toadstool at the episode's end was completely cut, without even an instrumental replacement.
Though DVD releases of "Kootie Pie Rocks" generally censor the aforementioned Milli Vanilli references, the PAL "Do the Koopa" DVD retains Milli Vanilli's original songs if the language is switched to Dutch.