The enzyme responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes electroneutral exchange of exoplasmic K+ for cytoplasmic H3O+. The molecular mechanisms of the catalytic exchange reaction are imperfectly understood, and await clarification of the precise topology of the enzyme with respect to the secretory membrane. Antibodies directed against H,K-ATPase subunits have been useful in confirming hydropathy plot predictions of HK alpha and HK beta secondary structure. The monoclonal antibody HK 12.18, which labels gastric mucosal parietal cells by immunocytochemistry, and which binds to a single M-r similar to 94,000 polypeptide by SDS-PAGE immunoblot of gastric microsomes, has been widely used as a specific marker of parietal cells in clinical and cell biological studies of acid secretion, and as a specific HK alpha probe in biochemical studies. However, the uncertain location of the HK 12.18 epitope has limited the antibody's usefulness as a topology probe. In this study, HM 12.18 immune reactivity with native H,K-ATPase tryptic peptides, HK alpha cDNA fragments expressed in bacteria, and overlapping synthetic HK alpha tridecapeptides, was used to identify the HK 12.18 epitope as seven consecutive amino acids (Asp(682)-Met-Asp-Pro-Ser-Glu-Leu(688)) in the cytoplasmic middle third of HK alpha.