We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ 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 the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.