A novel, simple, and rapid preparative method for purification of rat liver H(+)-ATP synthase by anion-exchange HPLC was developed. The H(+)-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H(+)-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-Pi exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H(+)-ATP synthase. An immunochemical study and a labeling experiment with N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD) demonstrated the presence of chargerin II ( a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the delta subunit in F1 were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit delta, and subunit epsilon. This is the first report of the existence of subunit b, factor 6, and chargerin II in H(+)-ATP synthase purified from rat liver mitochondria.