The mechanisms of mitochondrial proton (H⁺) leak under various pathophysiological conditions are poorly understood. In the present study it was hypothesized that different mechanisms underlie H⁺ leak in cardiac IR (ischaemia/reperfusion) injury and IPC (ischaemic preconditioning). Potential H⁺ leak mechanisms examined were UCPs (uncoupling proteins), allosteric activation of the ANT (adenine nucleotide translocase) by AMP, or the PT (permeability transition) pore. Mitochondria isolated from perfused rat hearts that were subjected to IPC exhibited a greater H⁺ leak than did controls (202±27%, P<0.005), and this increased leakage was completely abolished by the UCP inhibitor, GDP, or the ANT inhibitor, CAT (carboxyattractyloside). Mitochondria from hearts subjected to IR injury exhibited a much greater amount of H⁺ leak than did controls (411±28%, P<0.001). The increased leakage after IR was weakly inhibited by GDP, but was inhibited, >50%, by carboxyattractyloside. In addition, it was inhibited by cardioprotective treatment strategies including pre-IR perfusion with the PT pore inhibitors cyclosporin A or sanglifehrin A, the adenylate kinase inhibitor, AP₅A (diadenosine pentaphosphate), or IPC. Together these data suggest that the small increase in H⁺ leak in IPC is mediated by UCPs, while the large increase in H⁺ leak in IR is mediated by the ANT. Furthermore, under all conditions studied, in situ myocardial O₂ efficiency was correlated with isolated mitochondrial H⁺ leak (r²=0.71). In conclusion, these data suggest that the modulation of H⁺ leak may have important implications for the outcome of IR injury.