Murine autoimmune gastritis, induced by neonatal thymectomy, bears a striking similarity in pathology to the human autoimmune disease, pernicious anemia. Autoantibodies to parietal cells are found in both murine and human diseases. Monoclonal immunoglobulin G autoantibodies, obtained from neonatally thymectomized mice, have previously been shown to recognize two groups of gastric parietal cell antigens. In the present study, it is shown that two of these monoclonal autoantibodies, designated 1H9 and 2B6, are directed against the α subunit and β subunit, respectively, of the gastric hydrogen-potassium-stimulated adenosine triphosphatase (H+,K+-ATPase; proton pump). Monoclonal antibody 1H9 showed reactivity by immunoblotting with a 95-kilodalton component of dog gastric tubulovesicular membranes and with a fusion protein containing the hydrophilic domain of the α subunit of the H+,K+-ATPase. Monoclonal antibody 2B6 reacted by immunoblotting with the 60-90-kilodalton glycoprotein (β subunit) of the tomato lectin-purified dog H+,K+-ATPase and with the 60-90-kilodalton autoantigen purified with human parietal cell autoantibodies. Monoclonal antibody 2B6 also reacted with the deglycosylated 35-kilodalton core protein of the tomato lectin-purified 60-90-kilodalton β subunit and of the purified 60-90-kilodalton autoantigen. Parietal cell autoantibody-positive sera from 20 mice with experimentally induced gastritis showed reactivity predominantly with the α and/or β subunit of the gastric H+,K+-ATPase. Therefore, it is concluded that the major molecules targeted by parietal cell autoantibodies from mice with neonatal thymectomy-induced murine autoimmune gastritis and from humans with pernicious anemia are identical.