Cross-linking and two-dimensional crystallization studies have suggested that the membrane-bound gastric H,K-ATPase might be a dimeric alpha,beta-heterodimer. Effects of an oligomeric structure on the characteristics of E(1), E(2), and phosphoenzyme conformations were examined by measuring binding stoichiometries of acid-stable phosphorylation (EP) from [gamma-(32)P]ATP or (32)P(i) or of binding of [gamma-(32)P]ATP and of a K(+)-competitive imidazonaphthyridine (INT) inhibitor to an enzyme preparation containing approximately 5 nmol of ATPase/mg of protein. At <10 microM MgATP, E(1)[ATP].Mg.(H(+)):E(2) is formed at a high-affinity site, and is then converted to E(1)P.Mg.(H(+)):E(2) and then to E(2)P.Mg:E(1) with luminal proton extrusion. Maximal acid-stable phosphorylation yielded 2.65 nmol/mg of protein. Luminal K(+)-dependent dephosphorylation returns this conformation to the E(1) form. At high MgATP concentrations (>0.1 mM), the oligomer forms E(2)P.Mg:E(1)[ATP].Mg.(H(+)). The sum of the levels of maximal EP formation and ATP binding was 5.3 nmol/mg. The maximal amount of [(3)H]INT bound was 2.6 nmol/mg in the presence of MgATP, Mg(2+), Mg-P(i), or Mg-vanadate with complete inhibition of activity. K(+) displaced INT only in nigericin-treated vesicles, and thus, INT binds to the luminal surface of the E(2) form. INT-bound enzyme also formed 2.6 nmol of EP/mg at high ATP concentrations by formation of E(2).Mg.(INT)(exo):E(1)[ATP].Mg.(H(+)) which is converted to E(2).Mg.(INT)(exo):E(1)P.Mg.(H(+))(cyto), but this E(1)P form was K(+)-insensitive. Binding of the inhibitor fixes half the oligomer in the E(2) form with full inhibition of activity, while the other half of the oligomer is able to form E(1)P only when the inhibitor is bound. It appears that the catalytic subunits of the oligomer during turnover in intact gastric vesicles are restricted to a reciprocal E(1):E(2) configuration.