Concentrations of rhein and nitrofurantoin in the micromolar range induce Ca2+ release and the development of increased inner membrane permeability in liver mitochondria. Both compounds inhibit the mitochondrial glutathione reductase causing a depletion of GSH and an accumulation of GSSG in energized mitochondria. Under these conditions, the compounds also alter the oxidation state of pyridine nucleotides, NADH becoming oxidized while NADPH remains reduced. Using rhein or nitrofurantoin, together with t-butyl-hydroperoxide and beta-hydroxybutyrate, it is possible to selectively alter the NAD/NADH, the NADP/NADPH, and the GSSG/GSH ratios and to determine the effect of these different states on the ability of Ca2+ to produce a permeable inner membrane. No correlation between pyridine nucleotide ratios and sensitivity to Ca2+ was observed. Mitochondria are stable to Ca2+ when the GSH content is high, but become permeable when Ca2+ is present and GSH is converted to GSSG. It is proposed that the GSSG/GSH ratio, by controlling the reduction state of critical sulfhydryl groups, regulates lysophospholipid acyltransferase activity and, therefore, the ability of mitochondria to remain impermeable upon activation of the intramitochondrial Ca2+ requiring phospholipase A2.