1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP-38 or PACAP-27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP-38 and PACAP-27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single beta-cells, monitored by dual-wavelength fura-2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in beta-cells. PACAP antiserum also neutralized the [Ca2+]i-increasing action of synthetic PACAP-38 and PACAP-27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca2+]i in beta-cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from beta-cells, increased [Ca2+]i in beta-cells at both and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP-38 were detected in islets and a beta-cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7. [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive beta-cells, many of which were recruited by PACAP-38 into [Ca2+]i responses. 8. These results indicate that PACAP-38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses beta-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.