The insulin resistance syndrome is associated with atherosclerosis and cardiovascular events; however, the underlying mechanism of vascular dysfunction is unknown. The purpose of the current study was to assess endothelium-and smooth-muscle-mediated vasodilation in isolated coronary arteries from insulin-resistant rats and to determine whether insulin resistance alters the activity of the specific endothelium-derived relaxing factors. Male Sprague-Dawley rats were randomized to insulin resistance or control. Insulin resistance was induced by a fructose-rich diet. After 4 weeks of diet, coronary arteries were removed and vascular function was assessed in vitro using videomicroscopy. Acetylcholine (10–9–3× 10–5 M)-or sodium-nitroprusside (10–9–3× 10–4 M)-induced relaxations were determined. To evaluate the role of the specific endothelium-derived relaxing factors, several inhibitors were used, including N-nitro-L-arginine (LNNA), charybdotoxin/apamin (CTX/apamin), and indomethacin. Studies with nitroprusside showed that smooth-muscle-dependent relaxation did not differ between insulin resistance and control groups. In contrast, maximal relaxation (E max) to acetylcholine was decreased in the insulin resistance group (56±7%) versus control (93±3%). LNNA pretreatment further impaired E max in the IR group from 56±7 to 17±2%(p< 0.01). In control, E max was only slightly impaired by LNNA (93±3 to 63±6%; p< 0.05). The addition of CTX/apamin also decreased relaxation in the control group (93±3 to 47±7%; p< 0.05), whereas relaxation in insulin-resistant rats was not affected (45±5% with CTX/apamin vs. 56±7% with acetylcholine alone, NS). Pretreatment with indomethacin did not affect relaxation in either group, while pretreatment with the combination of LNNA and CTX/apamin completely abolished relaxation in both groups. Endothelium-dependent relaxation is impaired in small coronary arteries from insulin-resistant rats. The mechanism of this defect is related to a decrease in an endothelium-dependent, nitric oxide/prostanoid-independent relaxing factor or endothelium-derived hyperpolarizing factor.