Islet β-cells death during the development of Type I diabetes mellitus (IDDM) is suggested to be mediated by massive NO secretion, induced by cytokines in inter-islet macrophages and endothelium (Corbett et al. J.Clin.-Invest. 90.1992; Kolbet et al Diabetologia, 35, 1992). Treatment of the streptozotocin-induced diabetic animals with nitric oxide synthase (NOS) inhibitors attenuated hyperglycemia (Lukie et al BBRC.178.1991). On the other hand, the impaired EDRF/NO generation by endothelium of patients with IDDM (Calver et al J.Clin.Invest.90.1992) and in diabetic animals (Abiru et al Life sci 53, 1993) was found. We estimated the influence of insulin on NO generation and iNOS gene expression by rat macrophages and vascular smooth muscle cells in vitro. Rat peritoneal macrophages (10 8 cells/ml) stabilized for 24 hours in Dubelcco medium, or smooth muscle cells, were incubated with insulin (10-100μU/ml) without or in the presence of LPS (100ng/ml) for 30 min or 16 hours at 37°C. The generation of NO was measured according to Bredt and Snyder, (Proc.Natl. Acad Sci.87.1990) by the conversion of H 3 -L-arginine to citrulline. Induction of iNOS was analyzed by mRNA level for iNOS using RT-PCR and anti-sense primer. Insulin did not increase the NO generation after 30 min of preincubation. The about three time increase (from 5.91pmol/mg prot. to 16.3p-mol/mg prot) of H 3 L-citrulline formation was observed after 16h coincubation of insulin with macrophages. LPS itself was the weak activator of NO generation after the long incubation, and completely prevented the induction of iNOS mediated by insulin. We conclude that the increased iNOS activity by hyperinsulinemia may contribute to the auto-immune destruction of islet β-cells and microangiopaty.