Mitochondria can regenerate ascorbic acid from its oxidized forms, which may help to maintain the vitamin both in mitochondria and in the cytoplasm. In this work, we sought to determine the site and mechanism of mitochondrial ascorbate recycling from dehydroascorbic acid. Rat skeletal muscle mitochondria incubated for 3 h at 37 degrees C with 500 microM dehydroascorbic acid and energy substrates maintained ascorbate concentrations more than twice those observed in the absence of substrate. Succinate-dependent mitochondrial reduction of dehydroascorbic acid was blocked by inhibitors of mitochondrial Complexes II and III. Neither cytochrome c nor the outer mitochondrial membrane were necessary for the effect. The ascorbate radical was generated by mitochondria during treatment with dehydroascorbic acid and was abolished by ferricyanide, which does not penetrate the mitochondrial inner membrane. Together, these results show that energy substrate-dependent ascorbate recycling from dehydroascorbic acid involves an externally exposed portion of mitochondrial complex III.