Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase alpha subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercier et al. (1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the C-terminus of H,K-ATPase alpha subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitope for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888-907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM alpha helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.