The mitochondrial electrochemical gradient (Δp), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (^·O₂ ⁻) and H₂O₂ overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced ^·O₂ ⁻ and H₂O₂ production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased ^·O₂ ⁻ and H₂O₂ production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial ^·O₂ ⁻ and H₂O₂ generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced ^·O₂ ⁻ and H₂O₂ generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K⁺ ionophore used to dissipate ΔΨ, drastically diminished ^·O₂ ⁻ and H₂O₂ generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also contributed to ^·O₂ ⁻ generation by normal mitochondria and that IR mediated ΔΨ impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ΔpH restricts electron flow, controls a limited amount of ^·O₂ ⁻ generation, and results in a more reduced myocardium; however, IR causes ΔpH impairment and prompts a more oxidized myocardium.