Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation, however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 μM rotenone and antimycin) and by opening mitochondrial K_ATP channels with 100 μM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% & 38%, respectively. This effect of rotenone is not prevented by inhibiting Nox oxidase activation or scavenging superoxide with Peg-SOD, however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Since inhibition of ERK MAP kinase (10 μM PD98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U46619 also attenuated relaxation to hypoxia. Thus, coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.