Fragments of adult rat pancreas were incubated in vitro with tritiated serotonin at concentrations from 10(-8) to 10(-7) M. The pancreas exhibited an uptake of serotonin which was saturable, with an uptake constant (Km) of 8.75 x 10(-7) M, and a Vmax of 873 pmoles per gram. Specificity was determined by the addition of fluoxetine or norepinephrine to the reaction mixture, both at 10(-5) M. Fluoxetine significantly reduced the 3H-5HT uptake, whereas norepinephrine was without effect. Metergoline (10(-6) M), a specific 5-HT postsynaptic receptor blocker, similarly had no effect on the serotonin uptake in the pancreas. Radioautography of the fragments following uptake of tritiated serotonin (5 x 10(-8) M) revealed silver- grain aggregates dispersed along blood vessels in the interstitial spaces of the exocrine and endocrine pancreas, areas known to be traversed by nerve fibers. There were no silver- grain aggregates over the exocrine or islet parenchymal cells. These data support the hypothesis that the pancreas is innervated by serotonergic fibers. Further evidence for this hypothesis was provided by a preliminary study demonstrating the presence of tryptophan hydroxylase in pancreatic homogenates. These serotonergic fibers may be involved in the regulation of pancreatic secretion.