To examine the effects of chronic hyperinsulinemia on vascular tissues, we examined the production of superoxide anion (O(-2)) in the aortic tissues of control and exogenously hyperinsulinemic rats performed by the implantation of an insulin pellet for 4 wk. O(-2) production by aortic segments from hyperinsulinemic rats was 2. 4-fold (lucigenin chemiluminescence method) and 1.7-fold (cytochrome c method) of that of control rats without any differences in O(-2) degrading activities in aortic tissues, respectively (P < 0.025). The increment was completely abolished in the presence of either 100 micromol/l apocynin (an inhibitor of NADPH oxidase) or 10 micromol/l diphenyleneiodonium (an inhibitor of flavin-containing enzyme) and was exclusively endothelium dependent. Consistently, NAD(P)H oxidase activities in endothelial homogenate in hyperinsulinemic rats were dose dependently stimulated above the values of control rats, although these activities in nonendothelial homogenate were not significantly stimulated by insulin. Furthermore, an insulin effect was also demonstrated 1 h after exposing aortic tissues to insulin. These results indicate that O(-2) production specifically increases in endothelium of aortic tissues in chronic hyperinsulinemic rats through the activation of NAD(P)H oxidase.