Insulin secretion from pancreatic islets is controlled by peptides as well as by nutrients. We report here a novel, extraordinarily potent peptidergic regulation of insulin secretion. A 27-residue form of pituitary adenylate cyclase activating polypeptide (PACAP27) as low as 10(-14) to 10(-13) M stimulated insulin release from rat islets in a glucose-dependent manner. PACAP27 also increased cytosolic free Ca2+ concentration ([Ca2+]i) in islet beta-cells. Nitrendipine, a blocker of the L-type Ca2+ channel, abolished both [Ca2+]i and insulin responses. Vasoactive intestinal peptide, a peptide exhibiting 68% amino acid homology with PACAP, also increased [Ca2+]i in beta-cells but only at concentrations in the nanomolar range, indicating that PACAP27 is 4 logs more potent. A 38-residue form of the peptide (PACAP38) stimulated insulin release and increased beta-cell [Ca2+]i in a manner similar to that of PACAP27. PACAP-like immunoreactivity was demonstrated in pancreatic nerve fibers, islets, and capillaries. The results indicate that PACAP is a physiologically occurring peptide in pancreas and that PACAP, in a glucose-dependent manner, activates beta-cells presumably via a high affinity PACAP-selective receptor, raises [Ca2+]i by increasing the activity of L-type Ca2+ channels, and consequently stimulates insulin release. PACAP appears to be by far the most potent insulinotropic peptide known.