SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a] pyridine-3-acetonitrile) is an effective inhibitor of acid secretion in vivo and is a reversible, K+-competitive inhibitor of the gastric (H+ + K+)-ATPase in vitro. The actions of SCH 28080 have been studied on gastric vesicle preparations containing the (H+ + K+)-ATPase. At pH 7, inhibition was competitive with respect to K+ for both ATPase (Ki = 24 nM) and pNPPase (Ki = 275 nM) activities. A close analogue of SCH 28080 (methylated in the 1-N position), that was not expected to cross membranes freely, inhibited ATPase and pNPPase activity less effectively in intact vesicle preparations, where the lumenal (extracellular) face of the membrane was not directly accessible. This suggested that SCH 28080 inhibited both enzyme activities at a lumenal site on the enzyme. Being a protonatable weak base (pKa = 5.6), SCH 28080 would be expected to accumulate on the lumenal, acidic side of the parietal cell membrane in its protonated form. The potency of SCH 28080, relative to that of the "non-protonatable" analogue, increased at low pH, commensurate with the proportion of SCH 28080 in the protonated form. Thus the accumulating protonated form was the active inhibitory species. SCH 28080 (50 nM) blocked the rapid, K+-stimulated dephosphorylation of the catalytic phosphoenzyme intermediate of the (H+ + K+)-ATPase at room temperature. At 4 degrees, higher concentrations of the inhibitor were required, suggesting that the rate of inhibitor binding was slow at low temperatures.