Hyperinsulinemia is a risk factor for hypertension and arteriosclerosis. The mechanism by which it contributes to disease progression is not known. The present study examines the effects of insulin on endothelium-derived relaxing factors. Segments of rat mesenteric arterioles and aorta were set up for isometric recordings. The effect of insulin (1 mU/mL) on acetylcholine responses was examined with and without nitro-l-arginine, indomethacin, KCl (40 mmol/L), and apamin+charybdotoxin. Incubation with insulin (maximum response to acetylcholine 90.9±8.7% versus 90.7±4.5% for before versus after insulin, respectively), nitro-l-arginine, indomethacin, or high K+alone had no effect on these responses in mesenteric arterioles. Apamin+charybdotoxin significantly blunted responses to acetylcholine. When coincubated with nitro-l-arginine but not with indomethacin or high K+, insulin blunted the maximum response to acetylcholine (from 84.8±8.2% to 40.7±10.2% for before versus after insulin, respectively;P<0.01). When coincubated with apamin+charybdotoxin, insulin had no further effect. Coadministration of indomethacin with nitro-l-arginine had no greater effect than did nitro-l-arginine alone. The addition of insulin, together with nitro-l-arginine and indomethacin, significantly decreased the maximal response to acetylcholine from 96.6±5.3% to 52.9±10.8% (P<0.01). In the aorta, nitro-l-arginine abolished acetylcholine responses. Coadministration with insulin had no further effect. We conclude that insulin attenuates acetylcholine responses mediated by endothelium-derived hyperpolarizing factor in small but not large arteries. This effect of insulin is apparent only when NO is blocked and may be important in the development of hypertension or arteriosclerosis when reduced NO function has been reported.