Monoclonal antibody HK4013 raised against hog gastric vesicles dose-dependently inhibited gastric H+,K(+)-ATPase activity, formation of phosphoenzyme from ATP, and proton uptake into gastric vesicles. This antibody did not cross-react with related P-type ATPases such as hog kidney Na+,K(+)-ATPase or rabbit sarcoplasmic reticulum Ca(2+)-ATPase. It did not bind to the solubilized gastric H+,K(+)-ATPase, indicating that this antibody recognizes a higher-order structural epitope. The epitope is present on the cytosolic surface of H+,K(+)-ATPase. The addition of K+ to a solution containing gastric H+,K(+)-ATPase decreased the fluorescence intensity of the enzyme labeled with fluorescein isothiocyanate (FITC), showing the conformational transition from the E1 to E2K+ form. When H+,K(+)-ATPase was preincubated with HK4013, the addition of K+ did not decrease but increased the FITC fluorescence, indicating that this antibody changed the conformational state, at least near the ATP binding site. This is in contrast with the case of monoclonal antibody HK4001, which inhibited the decrease of the fluorescence. The fact that these two antibodies recognize different epitopes is consistent with previously reported facts that monoclonal antibody HK4001 inhibits the ouabain-insensitive K(+)-ATPase activity of rat distal colon but antibody HK4013 does not, and that the former stains the rabbit distal H+,K(+)-ATPase but the latter does not.