By immunoscreening of a cDNA expression library of rat pancreas with a polyspecific antibody to purified rat zymogen granule membranes, we have cloned a cDNA coding for a novel protein of about 16 kDa (ZG-16p). By both immunocytochemistry and Western blot analysis of different fractions of rat pancreas with anti-ZG-16 antibodies, the protein could be localized in the content fraction of zymogen granules and also, to a lesser extent, bound to the granule membranes. Computer-based sequence analysis revealed no significant homologies to any of the known proteins of zymogen granules. A N-terminal portion of about 20 amino acids was predicted as a potential secretory signal sequence and may reflect the intracellular localization of the protein. As revealed by Northern blot analysis of total RNA from various organs of the rat, expression of the corresponding gene is restricted by only pancreas, colon, duodenum, and, to a much lesser extent, stomach. No traces of ZG-16 RNA were detectable in any of the other tissues tested so far. Expression of the ZG-16-gene in pancreatic cells is slightly stimulated by treatment of rats with cerulein, a decapeptide analogue of cholecystokinin, which is known to stimulate secretion in acinar cells. In contrast, treatment of the rat pancreatic tumor cell line, AR4-2J, with 10 nM dexamethasone, which has been shown to increase the synthesis and secretion of all secretory enzymes of rat pancreas accompanied with an increase in the secretory machinery, leads to a remarkable increase in the expression of ZG-16. Thus the expression pattern of the ZG-16 gene in response to hormonal stimulation of rat pancreatic acinar cells resembles those found for most of the secretory enzymes. The localization of ZG-16p and the regulation of ZG-16 gene expression in response to hormonal stimulation of pancreatic acinar cells leads us to presume that this novel protein has a functional role in the complex and ill-understood processes involved in the regulated secretory pathway of these cells.