The role of islet constitutive nitric oxide synthase (cNOS) in insulin releasing mechanisms is highly controversial. By measuring enzyme activities and protein expression of NOS isoforms i.e. cNOS and iNOS (inducible NOS) in islets of Langerhans in relation to insulin secretion we now show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived NO strongly inhibits glucose-stimulated insulin release, presumably reflecting a negative feedback mechanism on the secretory machinery, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release. In contrast, addition of different NOS-inhibitors brought about a potentiation. These effects were evident also in K⁺-depolarized islets in the presence of the K⁺_ATP-channel opener diazoxide. Further, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, since certain concentrations of certain NOS inhibitors in contrast to the expected inhibitory effect, might indeed stimulate islet NO production. This is shown here by the observation that 0.5 mmol/l of the NOS inhibitor N^G-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets, and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might possibly be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type 2 non-insulin-dependent diabetes mellitus (NIDDM).