Stimulation of HCl secretion by gastric parietal cells requires the fusion of cytoplasmic H+-K+-ATPase-bearing tubulovesicles with the apical membrane. This insertion of membrane results in a dramatic increase in apical surface area through the formation of microvilli. To elucidate the elements that may stabilize the newly inserted H+-K+-ATPase within the apical membrane, we searched for specific cytoskeletal proteins associating with the gastric enzyme. We document by immunoblot analysis that ankyrin, spectrin, and actin copurify with H+-K+-ATPase microsomes prepared from gastric parietal cells. Coprecipitation of 125I-labeled native erythrocyte ankyrin with the H+-K+-ATPase from gastric microsomes using anti-H+-K+-ATPase antibodies suggests that ankyrin associates with the H+-K+-ATPase. Indirect immunofluorescence and confocal microscopy show that ankyrin and H+-K+- ATPase cosegregate within resting and secreting parietal cells. Taken together, these data suggest that the association of the gastric H+-K+- ATPase with spectrin and actin is mediated by ankyrin and that this interaction contributes to the maintenance of the polarized distribution of the enzyme to the apical domain of gastric parietal cells during acid secretion.