To localize binding sites for peptide YY (PYY) in the pancreas we utilized a slide-mount autoradiographic technique on frozen sections of rat pancreas incubated with 125I-Tyr36-PPY. Saturable autoradiographic labeling was located over pancreatic blood vessels, whereas islets, acinar cells, ducts, and neural elements did not appear to be specifically labeled. Isolated vascular fragments were prepared by collagenase digestion of rat pancreas. Binding experiments with 125I-Tyr36-PYY showed saturable binding to the fraction enriched in blood vessels but not to acini. Inhibition of 125I-Tyr36-PYY binding by nonradioactive neuropeptide Y (NPY) and PYY were similar, with half-maximal inhibition at 31.2 +/- 5 pM (n = 6); the potency of pancreatic polypeptide (PP) was 10,000 times lower. The binding site was classified as belonging to a Y1 type of NPY and/or PYY receptors, since [Leu31,Pro34]NPY, a specific Y1-receptor agonist, inhibited binding similar to NPY. To further localize the bound [125I-Tyr36]PYY within the blood vessels, light- and electron-microscopic autoradiographs were prepared and quantitated. Autoradiographic grains were located predominantly over vascular smooth muscle cells, although saturable localization was also seen over endothelial cells. It is concluded that in the pancreas Y1 receptors are predominantly located on vascular smooth muscle cells.