The release of nitric oxide (NO) through the oxidation of L-arginine by endothelial NO synthase (eNOS) promotes relaxation of vascular smooth muscle and plays a pivotal role in the maintenance of vascular homeostasis. Since L-arginine is an exclusive substrate for eNOS, its availability can regulate NO formation. Endothelial dysfunction, due to decreased NO function, is a prominent feature of obesity and type 2 diabetes and contributes to cardiovascular pathology. While eNOS expression is unchanged, vascular NO levels are decreased and L-arginine administration lowers blood pressure in patients with type 2 diabetes. This study tests the hypothesis that L-arginine restores coronary arterial endothelial function in obese Zucker rats (ZR), experimental models of type 2 diabetes. Male obese ZR (548 ± 12 g) and lean Sprague-Dawley (SD) rats (300 g) were used for the study. Mean arterial blood pressure (154 ± 3 mm Hg) and fasting blood glucose levels (186 ± 7 mg/dL) were elevated in obese ZR. Experiments were performed on isolated pressurized (80 mm Hg no flow conditions) small septal coronary arteries (200-300 μm) superfused with oxygenated (14% O2, 5% CO2 balanced with N2) Krebs buffer. Responses to an endothelium- and NO-dependent vasodilator, acetylcholine (1 nmol/L-3 μmol/L) were greatly attenuated in obese ZR coronary vessels (Δmax 5 ± 2 μm vs. 54 ± 10 μm). Acute in vitro pretreatment with the NOS substrate, 1 mmol/L L-arginine, enhanced acetylcholine-induced dilation and abolished the differences between obese ZR and SD arteries (Δmax 48 ± 18 μm vs. 61 ± 18 μm). These data show that blood pressure and blood glucose levels are elevated in obese ZR. Acetylcholine-induced endothelium-dependent vasodilation is greatly attenuated in small coronary arteries isolated from obese ZR. In addition, acute in vitro administration of L-arginine enhanced coronary endothelium-dependent responses and abolished the differences between obese ZR and SD coronary vessels. These results suggest that decreased L-arginine availability may contribute to coronary arterial endothelial dysfunction in obese ZR. Thus, L-arginine supplementation may present a novel therapeutic method to deter coronary vascular endothelial dysfunction and cardiovascular complications in patients with obesity and type 2 diabetes.