Gastric acid secretion involves the regulated recycling of the H, K-ATPase to and from the apical membrane of parietal cells. All of the steps in the regulated recycling of the H,K-ATPase should involve protein trafficking machinery. Based upon preliminary mass spectrometric analysis of isolated, H,K-ATPase-rich gastric microsomes, subunits of the retromer complex were identified. We sought to test the hypothesis that the retromer complex may play an important novel role in regulating the formation of tubulovesicular membranes in parietal cells and regulate the trafficking of the H, K-ATPase through this population of membranes. To begin to test this hypothesis, we characterized the retromer complex in gastric parietal cells. The mass spectrometry analysis was validated by Western blot of gastric microsomal membranes, immunoprecipitation, and immunofluorescent localization of retromer subunits. Moreover, we sought to characterize binding partners of the retromer by co-immunoprecipitation. Finally, we tried to develop a protocol for the in vitro assembly of the retromer complex. Taken together, the novel data obtained in this study support the presence of the retromer complex in a population of H,K-ATP-rich membranes in parietal cells. However, further study will be required to characterize the functional role of the retromer in parietal cell function.