By incubating the isolated rat myocardial mitochondria with xanthine-xanthine oxidase, an exogenous superoxide (O2.-) generating system, and by ischemia-reperfusion procedure of isolated rat heart as an endogenous O2.- generating system, it was found that both sources O2.- showed the same injurious effects on mitochondrial function resulting in (i) increasing proton leak rate, lowering proton pumping activity and H+/2e ratio of respiratory chain, and (ii) decreasing transmembrane potential of energized mitochondrial inner membrane by succinate oxidation. The injurious effects of O2.- on these mitochondrial bioenergitical parameters mentioned above exhibited a dosage- or reaction time-dependent mode. O2.- has no effects on the electron transfer activity and transmembrane potential of nonenergized mitochondria. Being a superoxide scavenger, 3,4-dihydroxylphenyl lactate showed obvious protection effects against damage of both exogenous superoxide sources from xanthine-xanthine oxidase system and endogenous O2.- source from ischemia-reperfused heart on proton pumping activity, H+/2e ratio and transmembrane potential of energized mitochondria. The experimental results presented here clearly showed that O2.- radicals induce an increase of proton leak in mitochondria directly related to the molecular mechanism of early phase damage of ischemia-reperfused heart, and also provide evidence to support our recent proposed hypothesis that electron leakage to oxygen to form O2.- in respiratory chain is one of the mechanical cause of proton leakage in mitochondria.