The present study was designed to examine the role of Ca2+in the regulation of digestive enzyme synthesis, to determine whether changes in intracellular Ca2+stores or cytosolic Ca2+caused the observed effects, and to establish the steps in the pathway of protein synthesis where the regulation occurs. Protein synthesis, polysome size, and the ratio of completed to nascent polypeptides were measured as a function of Ca2+in the intracellular stores and the cytoplasm of pancreatic acinar cells. Rat acini and rabbit pancreatic lobules were incubated in media containing 1 mMCaCl2with the following additives: cholecystokinin (CCK) octapeptide; the inhibitors of microsomal Ca2+ATPase, thapsigargin (THP) and 2,5-di(tertbutyl)-hydroquinone (BHQ); the intracellular Ca2+chelator, l,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA); an inhibitor of translational initiation, 7-methylguanosine 5'-triphosphate; and an inhibitor of translation elongation, cyclohexamide. THP and BHQ depleted intracellular pools of Ca2+and caused a sustained elevation in cytosolic [Ca2+]. Under these conditions, the polysome size diminished, and the ratio of completed proteins increased twofold relative to nascent polypeptides despite an overall decrease in net protein synthesis (55.3 ± 2.7% of control). These effects paralleled those caused by incubation with 1 nMCCK. Incubation of pancreatic acini with BAPTA plus THP or BHQ depleted the pool [Ca2+] without changing the cytosolic [Ca2+]. In addition, these agents decreased the net protein synthesis (30.1 ± 3.6% compared to control) and polysome size and increased the ratio of completed to nascent polypeptides to 2:1. These results suggest that depletion of intracellular stores of Ca2+without changes in cytosolic [Ca2+] decreases protein synthesis at translational initiation.