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Formation of H₂O₂ has been studied in rat heart mitochondria, pretreated with H₂O₂ and aminotriazole to lower their antioxidant capacity. It is shown that the rate of H₂O₂ formation by mitochondria oxidizing 6 mM succinate is inhibited by a protonophorous uncoupler, ADP and phosphate, malonate, rotenone and myxothiazol, and is stimulated by antimycin A. The effect of ADP is abolished by carboxyatractylate and oligomycin. Addition of uncoupler after rotenone induces further inhibition of H₂O₂ production. Inhibition of H₂O₂ formation by uncoupler, malonate and ADP+P_i is shown to be proportional to the ΔΨ decrease by these compounds. A threshold ΔΨ value is found, above which a very strong increase in H₂O₂ production takes place. This threshold slightly exceeds the state 3 ΔΨ level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169–202] that a high proton …