The gastric H,K-ATPase and the Na,K-ATPase both are stimulated by luminal K(+), but differ in sensitivity to K(+)-competitive inhibitors (ouabain and SCH28080), which implies a difference in structure near the luminal ion pathways in these two pumps. Knowledge of the amino acids in the H,K-ATPase that affect the mode of inhibition by SCH28080 and inhibitor affinity should provide insight into the regions of the membrane domain influencing the inhibitor selectivity and the luminal route to the ion transport site. Mutational scans in M4, 5, 6, and 8 have shown that amino acid residues affecting ion affinity (E343, K791, E795, E820, D824, E936) with either no or a lesser effect on the inhibitor affinity are located in the middle of the membrane domain. The residues significantly reducing inhibitor affinity, but not ion affinity (L809, P810, L811, T813, I816, Y925, T929), are located in the exoplasmic 5-6 loop and the luminal ends of M6 and M8. This suggests that the binding domain for SCH28080 contains the surface between L809 in the 5-6 loop and C813 at the luminal end of M6, approximately two helical turns out from the ion binding region, where it blocks an ion access pathway. The mutations that change inhibitor kinetics are on the opposing faces of M6 and M8 and apparently modify the normal ion pathway or, perhaps, create an alternate ion pathway.