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The generation of mitochondrial superoxide (O₂^−·) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H₂O₂ production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O₂^−· production by RET in isolated heart mitochondria. Measuring mitochondrial H₂O₂ production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O₂^−· production by RET responds to changes in O₂ concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O₂ …