Factors affecting the reduction of GSSG by rat liver mitochondria after a t-butyl hydroperoxide-induced (t-BOOH) oxidative stress were studied. The amounts of ADP and mitochondrial protein were adjusted to consume less than 50% of the available oxygen during the 8-min experimental period. A 4-min treatment of mitochondria with 24 nmol t-BOOH/mg protein (60 microM) oxidized 91% of total glutathione. In the presence of glutamate/malate, succinate or ascorbate/N,N,N',N'-tetramethyl-p- phenylenediamine (TMPD) (state 4 respiration), 84, 84, and 28% of the GSSG formed during t-BOOH treatment was reduced after 4 min, respectively. A similar extent of reduction was seen during state 3 respiration (1.5 mM ADP) with glutamate/malate, but no reduction occurred during state 3 respiration with either succinate or ascorbate/TMPD. The succinate-supported reduction of GSSG was completely blocked by rotenone, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). In contrast, oligomycin potentiated GSSG reduction using either glutamate/malate or succinate as substrates. Rotenone partially blocked glutamate/malate-supported GSSG reduction. NADPH levels were altered in direct proportion to the effects on GSSG reduction. The current data indicate that the reduction of GSSG in oxidatively-stressed isolated rat liver mitochondria occurs most efficiently during state 4 respiration and is independent of ATP synthesis. Both transhydrogenation and the transmembrane proton gradient appear to be important in NADPH regeneration and consequent GSSG reduction.