Aims: Angiotensin II (AngII)-induced superoxide (O₂^•−) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O₂^•− and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension. Results: Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O₂^•− production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O₂^•− and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O₂^•− production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK⁺_ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H₂O₂ in isolated mitochondria and diminished cytoplasmic O₂^•−. The mitoK⁺_ATP agonist diazoxide increased mitochondrial O₂^•−, cytoplasmic c-Src phosphorylation and cytoplasmic O₂^•− suggesting feed-forward regulation of cellular O₂^•− by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H₂O₂ scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O₂^•−, diminished aortic H₂O₂, and reduced blood pressure in hypertensive mice. Innovation and Conclusions: These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O₂^•− and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension. Antioxid. Redox Signal. 20, 281–294.