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Thomas was born in the rural community of Cayce, Mississippi, the son of a sharecropper. He moved with his family to Memphis, Tennessee, around 1920. His mother was a "church woman". Thomas made his debut as a performer at the age of six, playing a frog in a school theatrical production.

In 1960 he made his first recordings with his 17-year-old daughter Carla, for the Satellite label in Memphis, which changed its name to Stax the following year. The song, "Cause I Love You", featuring a rhythm borrowed from Jesse Hill's "Ooh Poo Pa Doo", was a regional hit; the musicians included Thomas' son Marvell on keyboards, Steinberg, and the 16-year-old Booker T. Jones. The record's success led to Stax gaining production and distribution deal with the much larger Atlantic Records.  Rufus Thomas continued to record for the label after Carla's record "Gee Whiz" reached the national R&B chart in 1961. He had his own hit with "The Dog", a song he had originally improvised in performance based on a Willie Mitchell bass line, complete with imitations of a barking dog. The 1963 follow-up, "Walking the Dog", engineered by Tom Dowd of Atlantic, became one of his most successful records, reaching #10 on the Billboard pop chart. He became the first, and still the only, father to debut in the Top 10 after his daughter had first appeared there. The song was recorded in early 1964 by the Rolling Stones on their debut album, and was a minor UK chart hit for Merseybeat group the Dennisons later that year.  As well as recording and appearing on radio and in clubs, Thomas continued to work as a boiler operator in the textile plant, where he claimed the noises sometimes suggested musical rhythms and lyrics to him, before he finally gave up the job in 1963, to focus on his role as a singer and entertainer. He recorded a series of novelty dance tracks, including "Can Your Monkey Do the Dog'" and '"Somebody Stole My Dog" for Stax, where he was often backed by Booker T. & the MGs or the Bar-Kays. He also became a mentor to younger Stax stars, giving advice on stage moves to performers like Otis Redding, who partnered daughter Carla on record.  After "Jump Back" in 1964, the hits dried up for several years, as Stax gave more attention to younger artists and musicians. However, in 1970 he had another big hit with "Do the Funky Chicken", which reached #5 on the R&B chart, #28 on the pop chart, and #18 in Britain where it was his only chart hit. Thomas improvised the song while performing with Willie Mitchell's band at a club in Covington, Tennessee, including a spoken word section that he regularly used as a shtick as a radio DJ: "Oh I feel so unnecessary - this is the kind of stuff that makes you feel like you wanna do something nasty, like waste some chicken gravy on your white shirt right down front." The recording was produced by Al Bell and Tom Nixon, and used the Bar-Kays, featuring guitarist Michael Toles. Thomas continued to work with Bell and Nixon as producers, and later in 1970 had his only number 1 R&B hit, and his highest pop charting record, with another dance song, "Do the Push and Pull". A further dance-oriented release in 1971, "The Breakdown", climbed to number 2 R&B and number 31 Pop. In 1972, he featured in the Wattstax concert, and he had several further, less successful, hits before Stax collapsed in 1976.
Rufus Thomas