Background. Superoxide (O 2 − • ) production by vascular NADPH oxidase is strongly implicated in the pathogenesis of hypertension; however, the role of mitochondrial O 2 − • is not clear. In this work we have investigated the role of mitochondrial O 2 − • in endothelial dysfunction and hypertension in cultured human aortic endothelial cells and in hypertensive mice using mitochondria-targeted superoxide dismutase mimetic mitoTEMPO, depletion or overexpression of mitochondrial superoxide dismutase (SOD2). Results. We found that supplementation of AngII-stimulated endothelial cells with mitoTEMPO decreased AngII-stimulated mitochondrial O 2 − • , inhibited production of cellular O 2 − • , restored nitric oxide (NO), attenuated c-Src activity and inhibited NADPH oxidase. SOD2 plasmid increased SOD activity in mitochondria but not in the cytoplasm, however, it inhibited cytoplasm NADPH oxidase and blocked AngII-induced cellular O 2 − • . SOD2 depletion with siRNA increased both basal and AngII-stimulated cellular O 2 − • due to higher NADPH oxidase activity while mitoTEMPO treatment mimicked SOD2 overexpression. Infusion of mitoTEMPO to AngII-treated mice attenuated AngII-induced hypertension. mito-TEMPO treatment after the onset of hypertension decreased blood pressure by 30 mm Hg both in AngII-infused and DOCA-salt mice (Table 1 ). The antihypertensive effect of mitoTEMPO was accompanied by decreased vascular O 2 − • , increased vascular NO production and recovery of endothelial-dependent relaxation. Interestingly, mice transgenic for mitochondrial SOD2 demonstrated attenuated AngII-induced hypertension and vascular oxidative stress similar to mitoTEMPO supplementation (Table 1 ). Conclusion. Our data show that mitochondrial O 2 − • is important for regulation of vascular NADPH oxidase and it represents a novel therapeutic target to treat endothelial dysfunction and hypertension. Table 1. Antihypertensive effect of mitoTEMPO (mT) and SOD2 overexpression (mm Hg) This research has received full or partial funding support from the American Heart Association, National Center.