In autoimmune gastritis antibodies against a membrane-bound parietal cell antigen of previously unknown function are present in the sera of patients. In this study, a vesicular membrane preparation of porcine gastric mucosa cells was found to be a potent antigenic source. This preparation blocked greater than 90% of antibody binding to a lysate of gastric mucosa cells. The membrane fraction contained H+,K+-ATPase (EC 188.8.131.52) as the major protein, which in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migrated with a weight of 92 kD. After reduction and alkylation, this component was resolved into two bands of similar staining intensity (92 and 88 kD). Immunoblotting analysis showed that sera of patients recognized antigen with pattern identical to the major protein of the vesicular membranes. Protein A-Sepharose beads preincubated with immunoglobulins of five individual patient (but not control) sera were all found to reduce both the H+,K+-ATPase activity and the amount of parietal cell antigen of a preparation of vesicular membranes solubilized in n-octylglucoside. Taken together, the results of this study indicate that the major parietal cell antigen is identical to the acid-producing enzyme, H+,K+-ATPase, of the parietal cell.