Superoxide anion can modulate vascular smooth muscle tone and is potentially involved in diabetic vascular complications. The present study was undertaken to characterize both vascular production and the enzymatic source of superoxide anion in type 2 diabetic rats. In the thoracic aorta of OLETF rats, endotheliumdependent relaxation was markedly attenuated compared with that of control (LETO) rats in association with a significant increase in superoxide production (2,421.39 ؎ 407.01 nmol ⅐ min -1 ⅐ mg -1 ). The increased production of superoxide anion was significantly attenuated by diphenyleneiodonium (DPI; 10 mol/l), an inhibitor of NAD(P)H oxidase. The production of superoxide anion in response to NADH as a substrate was markedly increased in the vascular homogenates, but NADPH, arachidonic acid, xanthine, and succinate produced only small increases in chemiluminescence. In line with these results, studies using various enzyme inhibitors, such as DPI, allopurinol, rotenone, N G -monomethyl-L-arginine, and indomethacin, suggest that the predominant source of superoxide anion in vascular particulate fraction is NADH-dependent membranebound oxidase. Furthermore, the expression of p22phox, a major component of vascular NAD(P)H oxidase, was markedly increased in the aorta from OLETF rats compared with that of LETO rats. These findings suggest that upregulated expression of p22phox mRNA and enhanced NADH oxidase activity contribute to the impaired endothelium-dependent vasodilation in OLETF rats. Diabetes 51:522-527, 2002