24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.