We have recently shown that permeabilization of the inner mitochondrial membrane by calcium plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates [Fagian, M. M., Pereira da Silva, L., Martins, I. S. and Vercesi, A. E. (1990) J. Biol. Chem. 265, 19955-19960]. In this study we show that mitochondria could regenerate and sustain a membrane potential (ΔΨ) comparable to the control experiment after the protein aggregates were cleaved by dithiothreitol. The addition of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid, which removes Ca 2+ but does not eliminate the protein aggregates, caused an incomplete and nonsustainable recovery of ΔΨ. Exogenous catalase prevented the disruption of membrane potential and decreased the production of membrane protein aggregates when mitochondria were incubated in the presence of Ca 2+ alone or Ca 2+ plus a prooxidant. This strongly indicates that H 2O 2 and possibly other H 2O 2-derived reactive oxygen species are involved in the mechanism of membrane protein aggregates production that may result in the process of membrane pore formation.