He performed with Bob Dylan in concert in 1965, including playing Hammond organ with Dylan at the Newport Folk Festival, and in the recording studio in 1965 and 1966. Kooper also played the Hammond organ riffs on Dylan's "Like a Rolling Stone". It was in those recording sessions that Kooper met and befriended Mike Bloomfield, whose guitar playing he admired. He worked extensively with Bloomfield for several years. Kooper played organ once again with Dylan during his 1981 world tour.
Kooper joined the Blues Project as their keyboardist in 1965; he left the band shortly before their gig at the Monterey Pop Festival in 1967, although he did play a solo set at the famous festival, as evidenced by bootlegs of the event. He formed Blood, Sweat & Tears in 1967, leaving due to creative differences in 1968, after the release of the group's first album, Child Is Father to the Man. He recorded Super Session with Bloomfield and Stills in 1968, and in 1969 he collaborated with 15-year-old guitarist Shuggie Otis on the album Kooper Session. In 1975 he produced the debut album by the Tubes.