The role nitric oxide (NO) plays in physiological insulin secretion has been controversial. Here we present evidence that exogenous NO stimulates insulin secretion, and that endogenous NO production occurs and is involved in the regulation of insulin release. Radioimmunoassay measurement of insulin release and a dynamic assay of exocytosis using the dye FM1-43 demonstrated that three different NO donors-hydroxylamine (HA), sodium nitroprusside, and 3-morpholinosydnonimine (SIN-1)-each stimulated a marked increase in insulin secretion from INS-1 cells. Pharmacological manipulation of the guanylate cyclase/guanosine 3',5'-cyclic monophosphate pathway indicated that this pathway was involved in mediating the effect of the intracellular NO donor, HA, which was used to simulate endogenous NO production. This effect was further characterized as involving membrane depolarization and intracellular Ca(2+) ([Ca(2+)](i)) elevation. SIN-1 application enhanced glucose-induced [Ca(2+)](i) responses in primary beta-cells and augmented insulin release from islets in a glucose-dependent manner. Real-time monitoring of NO using the NO-sensitive fluorescent dye, diaminofluorescein, was used to provide direct and dynamic imaging of NO generation within living beta-cells. This showed that endogenous NO production could be stimulated by elevation of [Ca(2+)](i) levels and by glucose in both INS-1 and primary rat beta-cells. Scavenging endogenously produced NO-attenuated glucose-stimulated insulin release from INS-1 cells and rat islets. Thus, the results indicated that applied NO is able to exert an insulinotropic effect, and implicated endogenously produced NO in the physiological regulation of insulin release.