In order to determine whether both insulin and C-peptide have an inhibitory action on insulin secretion, the isolated rat pancreas was perfused with exogenous rat insulin or synthetic rat C-peptide in the presence of high or low concentration of glucose. In the presence of 2.8 mM glucose, exogenous rat insulin (4 or 10 ng/ml) and C-peptide 1 and 2 (100 ng/ml) show no effect on the insulin levels in the outflow from the perfused pancreas. However, the insulin response to 16.7 mM glucose decreased in the presence of exogenous rat insulin or synthetic rat C-peptide, showing a biphasic pattern of glucose-induced insulin release. When rat insulin or C-peptide were added at 20 min and removed at 40 min while the isolated pancreas was exposed to a 60-min glucose infusion (16.7 mM), glucose-induced insulin secretion decreased during the infusion of rat insulin or C-peptide. The present study clearly showed that exogenous rat insulin and synthetic rat C-peptide 1 and 2 inhibited glucose-induced insulin secretion. Although the suppressive mechanisms of the exogenous insulin and the C-peptide on insulin release are not yet proved, the inhibitory process is considered to be related to cyclic AMP in the pancreatic B-cell.