The generation of mitochondrial superoxide (O 2 ˙̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H 2O 2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O 2 ˙̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H 2O 2 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 O 2 ˙̄ production by RET responds to changes in O 2 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 O 2 ˙̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O 2 ˙̄ production by RET. An analysis of O 2 ˙̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O 2 ˙̄ production in both pathological and physiological conditions. We conclude that O 2 ˙̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.