We have recently reported the cloning of the rat zymogen granule membrane glycoprotein GP-3 and the related pancreatic secretory lipase (Wishart, M. J., Andrews, P. C., Nichols, R., Blevins, G. T., Logsdon, C.D., and Williams, J. A. (1993) J. Biol. Chem. 268, 10303-10311). Specific antipeptide antibodies were generated against both GP-3 and secretory lipase and used for the biochemical and physiological characterization of GP-3. Western blotting confirmed that GP-3 was found exclusively in zymogen granule membranes and was absent from zymogen granule content which contains the majority of secretory lipase. Extraction of zymogen granule membranes with Triton X-114 showed GP-3 to be significantly more hydrophobic than lipase. The GP-3 amino acid sequence contains one potential N-linked glycosylation site at Asn-336. The loss of concanavalin A labeling after both chemical deglycosylation with trifluoromethanesulfonic acid and enzymatic deglycosylation with N-glycanase showed GP-3 to possess a small N-linked oligosaccharide side chain. Digestion of intact and permeabilized zymogen granules with the nonspecific protease Pronase localized GP-3 to the inner surface of zymogen granule membranes. Since GP-3 is resident on the inner surface of the zymogen granule membrane, it should appear on the outer cellular surface after exocytosis. Although membrane attachment of GP-3 was resistant to treatment with phosphatidylinositol-specific phospholipase C, we observed that GP-3 is released into the pancreatic juice and that secretion of GP-3 was greatly enhanced by cholecystokinin.