We hypothesized that neutralization of TNF- at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species (ROS). To test this, we employed a murine model of ischemia/reperfusion (I/R, 30 min/90 min) administering neutralizing antibodies at the time of reperfusion to block the actions of TNF-. Also, to decipher the sources of ROS stimulated by TNF- in I/R, we inhibited several enzymes involved in ROS production. TNF- expression (mRNA & protein) was elevated in I/R in mice without antibody; whereas, administration of anti-TNF- prior to reperfusion attenuated TNF- expression. Immunostaining revealed that TNF- was localized in vascular smooth muscle cells, mast cells and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production, but anti-TNF- at the onset of reperfusion partially restored NO-mediated coronary arteriolar dilation and reduced superoxide production. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. NAD(P)H oxidase activity, xanthine oxidase activity, and formation of nitrotyrosine residues were increased in untreated mice (compared to shams), but these variables were restored to control levels by anti-TNF- prior to reperfusion. Interestingly, neither endothelial function nor superoxide generation during I/R were significantly affected in neutropenic animals, or by the neutrophil NAD(P)H oxidase inhibitor, MFH244, suggesting that the effects of TNF- are not through neutrophil activation. These results suggest that myocardial ischemia initiated expression of TNF- inducing vascular oxidative stress, independent of neutrophil activation, leads to coronary endothelial dysfunction.