Integration into the cytoplasmic membrane and function of the three F0 subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible. This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits. AU three F0 subunits were found to be required for the establishment of efficient H+ conduction. Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not. The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N'-dicyclohexylcarbodiimide. Also ATP-dependent H+ translocation was not catalysed unless all three F0 subunits were present in the membrane. The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits.