In this study, we show that mitochondrial membrane permeability transition in Ca(2+)-loaded mitochondria treated with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or inorganic phosphate (P(i)) is preceded by enhanced production of H2O2. This production is inhibited either by ethylene glycobis(b-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or Mg2+, but not by cyclosporin A. Permeability transition is prevented either by EGTA, catalase or dithiothreitol, suggesting the involvement of Ca2+, H2O2 and oxidation of membrane protein thiols in this mechanism. When mitochondria are incubated under anaerobiosis, no permeabilization or H2O2 production occurs. Based on these results we conclude that mitochondrial permeability transition induced by P(i) or FCCP-uncoupling is dependent on mitochondrial-generated reactive oxygen species.