To test the capability of the pancreas to change the proportions of proteins secreted in response to acute stimulation, we examined the effect of cholecystokinin-pancreozymin stimulation on the relative discharge of 16 radiolabeled secretory proteins from the in vitro rabbit pancreatic gland. [35S]-methionine was added to the bathing medium 4 h prior to the experimental period. Secretion was then collected from the pancreatic duct for two 1-h basal periods and for two 20-min periods following stimulation with 5.1 mM cholecystokinin. Proteins were separated by two-dimensional isoelectric focusing/sodium dodecyl sulfate gradient gel electrophoresis, and the proportion of each radiolabeled protein in the secretion was determined in each period. Statistical analysis of the relative distribution of radioactive proteins in secretion showed an increased variability in the distribution of secreted proteins following stimulation (P < 0.005). Procarboxypeptidase A, a protein tentatively identified as an acidic form of chymotrypsinogen, and a basic form of chymotrypsinogen were significantly more stimulated than the average radiolabeled protein by 25, 31, and 17%, respectively, whereas the acidic form of trypsinogen was significantly less stimulated by 18%.