We have recently shown that ruthenium red, a non-competitive inhibitor of the mitochondrial Ca2+ uniporter, can reduce tert-butyl hydroperoxide via a Fenton-type reaction. In respiring mitochondrial preparations containing tert-butyl hydroperoxide, redox cycling of ruthenium red occurs and causes the amplification of methyl radical generation (Meinicke, A. R., Zavan, S. S., Ferreira, A. M. C., Vercesi, A. E., and Bechara, E. J. H. (1996) Arch. Biochem. Biophys. 328, 239-244). In this study we show that ruthenium red can act as an antioxidant preventing mitochondrial damage when the respiratory chain is reduced or when ascorbate is present. Ruthenium red can catalyze the degradation of hydrogen peroxide into H2O and O2. We show here that ruthenium red prevents both accumulation of mitochondrial generated H2O2 and swelling in the presence of the Ca2+ ionophore A23187. Under these conditions the damage induced by Ca2+ ions and either tert-butyl hydroperoxide or inorganic phosphate is promoted by mitochondrial-generated reactive oxygen species. Swelling induced by phenylarsine oxide, a thiol cross-linker, by a mechanism independent of free radicals is not inhibited by ruthenium red. These data provide evidence that the antioxidant behavior of ruthenium red under our conditions is due to its ability to destroy peroxides, which is related to its redox cycling and is prevalent over the Fenton-type reaction.