Two fractions of gastric mucosal membranes obtained by Ficoll sucrose density gradient centrifugation were studied by a variety of techniques to localize the polypeptides. Gel electrophoresis showed the presence of five major polypeptides and several minor ones. Only one of these, 82,000 daltons, was available for iodination in the intact tissue. The two membrane fractions differed in their accessibility to peroxidase. The denser fraction showed two major defined iodination peaks at 82,000 and 102,000 daltons. Freeze thawing and iodinating with 131I produced additional labeling of peaks as well as relabeling the 82,000 dalton component, showing it was accessible from both sides of the membrane. The two major components were also sensitive to cross linking, the 102,000 polypeptide being especially sensitive to -SH oxidation. Proteolysis with trypsin removed both components in the denser membrane fraction, in addition to inhibiting the K+ adenosine triphosphatase (ATPase) and K+ p nitrophenylphosphatase of that fraction. Phosphorylation with [γ 32P]ATP labeled the 102,000 dalton component and K+, HCO3- and p nitrophenylphosphate reduced the level of labeling. Hence the 102,000 region contains a subunit of the ATPase, is readily iodinated in inside out vesicles and is the most available for interpeptide S-S cross linking.