Activated macrophages release peroxynitrite anion (ONOO-), which has been recently shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. In this work, we report that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and fumarate reductase, are inactivated by biologically relevant concentrations of peroxynitrite. Enzyme inactivation was accompanied by a significant inhibition of succinate-dependent respiration in intact cells as well as in the membrane-rich fraction. Peroxynitrite also inhibited NADH-dependent oxygen consumption which depends almost exclusively on fumarate reductase activity in T. cruzi epimastigotes. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite-mediated inactivation of succinate dehydrogenase and fumarate reductase may be a key mechanism of macrophage-mediated cytotoxicity to T. cruzi, through inhibition of the energetic metabolism of the parasite.