Four methods were compared to purify F_o from porcine heart mitochondria. The best results were obtained by the following method: after removing F₁-ATPase with NaBr incubation from submitochondrial F_oF₁-ATPase, F_o was solubilized with CHAPS and purified by sucrose density gradient centrifugation. SDS-PAGE with silver staining showed about 85% purity of the isolated F_o and 9 different subunits including b, OSCP, d, a, e, F₆, IF₁, A6L and c. The purified F_o was then incorporated into asolectin liposomes, the reconstituted F_o showed higher H⁺ translocation activity and after F_o was reconstituted with F₁-ATPase, the resulted F_oF₁-ATPase complex exhibited high ATP hydrolysis activity and high sensitivity to oligomycin. The results provide evidence for successful purification, reconstitution of F_o with high H⁺ translocation activity and its relationship with phospholipids.