In March 1964, Hendrix recorded the two-part single "Testify" with the Isley Brothers. Released in June, it failed to chart. In May, he provided guitar instrumentation for the Don Covay song, "Mercy Mercy". Issued in August by Rosemart Records and distributed by Atlantic, the track reached number 35 on the Billboard chart.
Hendrix toured with the Isleys during much of 1964, but near the end of October, after growing tired of playing the same set every night, he left the band. Soon afterward, Hendrix joined Little Richard's touring band, the Upsetters. During a stop in Los Angeles in February 1965, he recorded his first and only single with Richard, "I Don't Know What You Got (But It's Got Me)", written by Don Covay and released by Vee-Jay Records. Richard's popularity was waning at the time, and the single peaked at number 92, where it remained for one week before dropping off the chart. Hendrix met singer Rosa Lee Brooks while staying at the Wilcox Hotel in Hollywood, and she invited him to participate in a recording session for her single, which included the Arthur Lee penned "My Diary" as the A-side, and "Utee" as the B-side. Hendrix played guitar on both tracks, which also included background vocals by Lee. The single failed to chart, but Hendrix and Lee began a friendship that lasted several years; Hendrix later became an ardent supporter of Lee's band, Love.
In July 1965, on Nashville's Channel 5 Night Train, Hendrix made his first television appearance. Performing in Little Richard's ensemble band, he backed up vocalists Buddy and Stacy on "Shotgun". The video recording of the show marks the earliest known footage of Hendrix performing. Richard and Hendrix often clashed over tardiness, wardrobe, and Hendrix's stage antics, and in late July, Richard's brother Robert fired him. He then briefly rejoined the Isley Brothers, and recorded a second single with them, "Move Over and Let Me Dance" backed with "Have You Ever Been Disappointed". Later that year, he joined a New York-based R&B band, Curtis Knight and the Squires, after meeting Knight in the lobby of a hotel where both men were staying.