We previously reported that tumor necrosis factor-alpha (TNF) contributes to endothelial dysfunction in the prediabetic Zucker obese rat. To determine if TNF plays a similar role in endothelial dysfunction in fully developed diabetes, we tested the hypothesis that impairment in coronary endothelial function in type II diabetes (Zucker diabetic fatty rat, ZDF) is due to the overexpression of TNF via TNF receptors (TNFRs). In ZDF rats, dilation (isolated arterioles) to endothelium-dependent vasodilator, acetylcholine (ACh), was blunted compared to lean rats, but dilation to the endothelium-independent vasodilator sodium nitroprusside (SNP) was comparable. Neutralizing antibodies to TNF (72 hr treatment; 24 mg/kg, i.p.) restored NO-mediated coronary arteriolar dilation in ZDF rats, but did not alter endothelium-dependent vasodilation in lean rats. Expression of TNFR1 (mRNA) was significantly increased in coronary arterioles in ZDF vs lean, but TNFR 2 mRNA expression was similar in the two groups. Western blotting shows the protein expression of TNF was higher in both heart and coronary arterioles in ZDF rats than that in lean rats. NAD(P)H oxidase activity and H2O2 production increased significantly in ZDF rats, but anti-TNF decreased both NAD(P)H oxidase activity and H2O2 production. These results demonstrate the endothelial dysfunction occurring in type II diabetes is the result of effects of the inflammatory cytokine TNF, mainly through the overexpression of TNF-R1, which subsequently activates NAD(P)H oxidase and H2O2.