The GP2/THP family of glycosyl phosphatidylinositol (GPI)-anchored proteins is targeted to apical secretory compartments in polarized epithelial cells. We demonstrate in the rat exocrine pancreas that enzyme-mediated release of GP2 from acinar cell membranes represents a pH-dependent process regulated by bicarbonate secreted from ductular cells. Release of GP2 from secretin-stimulated pancreatic lobules, which retain intralobular ducts, was inhibited by (i) bicarbonate substitution, (ii) chloride substitution, and (iii) DIDS, a potent inhibitor of chloride-bicarbonate exchange. These inhibitory effects were not observed in preparations of pancreatic acini devoid of ductal elements. Enzymatic cleavage of GP2 and amylase release from pancreatic acini varied directly as a function of pH of the acinar human. Alkali-induced GP2 release could be correlated with ultrastructural and biochemical evidence for stimulated retrieval (endocytosis) of exocytic membranes at the acinar lumen. Our study defines functional roles for ductal bicarbonate in acinar cell and lumen physiology and provides a potential explanation for the biological significance of enzyme-mediated cleavage of GP2 from the apical plasma membrane.