Gastric intracellular tubulovesicles fuse with the apical membrane upon histamine stimulation. Disulfide cross-linking of isolated gastric tubulovesicles by cupper-o-phenanthroline (CuP) opened the chloride channel in the vesicles. A functional monoclonal antibody raised against H+,K(+)-ATPase of gastric vesicles inhibited both the enzyme activity and the CuP-induced opening of the chloride channel. This fact indicates that the chloride channel is part of the function of the ATPase. Another evidence which supports the above concept that both the pump and the chloride channel coexist in the same molecule was obtained in this study. The conformation of H+,K(+)-ATPase was changed in the direction of E2 form by incubation with SCH 28080 or low concentrations of K+. SCH 28080 is an H+,K(+)-ATPase specific inhibitor and binds to the high affinity K+ site. Both SCH 28080 and K+ inhibited the channel opening, indicating that the channel opening by the S-S cross-linking depends on the conformational state of the enzyme.