Biliary excretion of glutathione disulfide (GSSG) is used as an index of oxidative stress. Analysis of endogenous thiols and disulfides in rat bile by reverse phase high performance liquid chromatography with electrochemical detection revealed an unknown disulfide which eluted immediately after GSSG. This disulfide was tentatively identified as the mixed disulfide of glutathione (GSH) and cysteinylglycine (Cys-Gly), based on its coelution on a reverse phase column with the synthetic GS-Cys-Gly. GS-Cys-Gly was also detected in bile of other species. On analyzing species differences in biliary excretion of GSH-related thiols and disulfides, it was concluded that biliary excretion of GS-Cys-Gly was related to the excretion of both GSSG and Cys-Gly, which is formed from GSH by gamma-glutamyltransferase (gamma-GT)-catalyzed hydrolysis. Species with low hepatic gamma-GT (i.e., hamsters and mice) excreted little Cys-Gly in bile. These animals excreted negligible amounts of GS-Cys-Gly even when biliary excretion of GSSG was markedly increased by paraquat-induced oxidative stress. Rats and guinea pigs, which have high hepatic gamma-GT activities, excreted large amounts of both Cys-Gly and GS-Cys-Gly. Treatment of rats with acivicin, an inhibitor of gamma-GT, decreased the biliary excretion of both Cys-Gly and GS-Cys-Gly. Paraquat treatment of rats resulted in an increase in GSSG excretion with concomitant increase of GS-Cys-Gly excretion. Rabbits, which also have high hepatic gamma-GT activity, excreted little GS-Cys-Gly into bile.(ABSTRACT TRUNCATED AT 250 WORDS)