The membrane F0 sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F,-depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin-sensitive proton conduction, or recombined with purified F, to give the oligomycin-sensitive F,F,-ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDSpolyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F,, polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD-binding protein, the nuclear encoded protein ) J. Mol. Biol. 197, 89-1001