Abstract The tetracycline resistance gene ( tetA) was cloned downstream of the lac promoter. When expression of the tetA gene in E. coli cells carrying the lac I q gene was induced with isopropyl β- d-thiogalactopyranoside, the tetracycline resistance protein (TetA) was overproduced, amounting to about 30% of the integral cytoplasmic membrane protein. Essentially pure TetA protein could be obtained by solubilization with 1.25% n- octyl-β- d-glucopyranoside and one-step purification by DEAE Sepharose CL-6B column chromatography. The TetA protein was incorporated into proteoliposomes with F 0F 1-ATPase. The proteoliposomes exhibited [ 3H]tetracycline transport dependent on ATP hydrolysis. The specific activity was about 2 nmol/mg protein/min. The proteoliposomes also showed H + efflux coupled with tetracycline influx. Tetracycline/H + antiport by proteoliposomes reconstituted with the Ser-65 → Cys mutant TetA protein was inhibited by N-ethylmaleimide. These results proved for the first time that the tetracycline/H + antiport is only mediated by the TetA protein.