Isolated rat liver mitochondria undergo extensive swelling and disruption of membrane potential when they accumulate Ca2+ in the presence of a prooxidant such as diamide or t-butylhydroperoxide. The phenothiazinic drug trifluoperazine, at concentrations (15-35 microM) which do not inhibit respiration or the influx of Ca2+ into mitochondria, significantly protected mitochondria against the deleterious effects of Ca2+ plus a prooxidant. In contrast, at concentrations higher than 100 microM the drug potentiated these deleterious effects of Ca2+ and prooxidants and had a damaging effect per se on the inner mitochondrial membrane. It is proposed that the protection conferred by the drug is mediated by changes in membrane protein structure that decrease the production of protein thiol cross-linkings which occur when mitochondria accumulate calcium under oxidant stress conditions.