Diabetes mellitus is a major cause of ischemic coronary artery disease. Endothelial dysfunction is implicated in the pathogenesis of diabetic vascular disease. To examine coronary blood flow (CBF) regulation with endothelium-derived nitric oxide (EDNO) in the diabetic state, we compared the effects of both acetylcholine (ACh) and adenosine (Ado) on left circumflex coronary artery (LCX) blood flow in 12 vehicle-treated and 21 dogs made diabetic with alloxan anesthetized with pentobarbital. All dogs were pretreated with aspirin to inhibit endogenous prostaglandins. None of the hemodynamic parameters were significantly different in the two groups. The percent change in coronary vascular resistance (CVR) after ACh (100 ng/kg) infusion was significantly attenuated in diabetic dogs (-56.5±1.4%) as compared with vehicle-treated dogs (-64.5±1.2%)(p< 0.01), whereas the effect of Ado (1 μg/kg) was not different between the two groups (-71.1±1.5% in vehicle,-67.0±1.3% in diabetes). After infusion of incremental doses of N G-nitro-L-arginine methyl ester (L-NAME) 10-5-10-3 M, the effect of ACh was progressively inhibited in both groups and was different no longer between the two groups after the maximal dose. L-Arginine (L-ARG), but not D-ARG, significantly restored the effect of ACh in diabetic dogs but did not affect vehicle-treated dogs. The effect of Ado did not change after L-and D-ARG administration. Cu, Zn-superoxide dismutase (Cu, Zn-SOD) had no effect on any of the effects of ACh and Ado in diabetic dogs. Regulation of CBF with EDNO is impaired in dogs with alloxan-induced diabetes, and this impairment is partially restored by L-ARG.