Oxidative stress, in particular superoxide anions, affect function of large vessel segments [Gorlach et al., 2000, Brandes et al., 2004]. This study addresses the functional relevance of NADPH oxidase for extracellular superoxide production and coronary flow regulation. As a model of NADPH oxidase inactivation, mice of a gp91phox-/- strain were used. Isolated hearts from C57/Bl6 and gp91phox-/- mice were perfused at a perfusion pressure of 80±3 mmHg and paced at 505±5 bpm. Coronary venous superoxide concentration was measured via oxidation product of spin probe N1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) and superoxide anions using electron paramagnetic resonance 1) under baseline conditions 2) endothelium-dependent vasodilation and 3) TNFa stimulation. Gp91phox-/- exhibited no decreased coronary venous superoxide concentrations under baseline conditions compared to wild-type mice. Coronary flow response as well as ventricular pressure development towards acetylcholine was more pronounced in gp91phox-/- compared to wild-type mice. During activation of NADPH oxidase by TNFa?an increased coronary venous superoxide concentration could be observed in wild-type but not in gp91phox-/- mice. These results suggest that subunit gp91phox is of little importance for baseline coronary flow control but may interact with endothelium- dependent coronary flow regulation.