Isolated purified parietal cells from rabbit gastric mucosae were used to investigate the functions of phosphoproteins associated with regulation of acid secretion. These cells responded well to secretagogues, increasing their level of acid secretion by ca. four-fold. Stimulation of acid secretion was accompanied by a distinct morphological change, which involves the cytoskeletal system of the parietal cell, particularly the microfilaments, as evidenced by potent inhibition of secretion by cytochalasin E. Parietal cells contained several actin binding proteins, at least one of which (47 kD) had a similar molecular weight as a previously identified stimulus-related phosphoprotein. A single 130 kD parietal cell protein was immunoreactive with anti-vinculin monoclonal antibody, correlating with another previously identified stimulus-related phosphoprotein. The functional role of these proteins may be to link the microfilaments with the secretory membrane, an important step in the generation and/or maintenance of the stimulated state of acid secretion in the parietal cell.