Eye of the Tiger
During the filming of 4.06 Yellow Fever, there is a scene where Dean is in the Impala, miming to Eye of the Tiger - a 1982 hit for the band Survivor. Sam approaches and taps on the roof of the Impala, interrupting Dean's singing. In one take of the scene, Jared decided not to give Jensen his cue to stop, and decided to see what would happen.
Luckily director Philip Sgriccia (who is also the show's post-production director and producer of each season's gag reel) kept the cameras rolling as Jensen spontaneously launched into a roof top performance. After positive response from Warner Bros., it was decided that rather than save the outtake just for the gag reel, it would air after the episode on October 23, 2008. Fans loved it, and the song has now become a standard at Convention karaoke nights and any fan gathering.
Jensen and music for more on Jensen's musical talents.