The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in uiuo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in uiuo, and during liver perfusions with BSPcontaining perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de nouo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in uiuo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de nouo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de nouo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-K, for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3 pmoVg): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels. (HEPATOLOGY 1995;21:1387-1394.) Glutathione (GSH) conjugation is an important biotransformation reaction of xenobiotics and endogenous compounds containing electrophilic groups. The reaction is catalyzed by the glutathione-S-transferases (Enzyme Committee 2.5.1.18) and requires the endogenous tripeptide GSH. We have studied the dependence of GSH conjugation on hepatic GSH in the rat in vivo Abbreviations: GSH, glutathione; BSP, bromosulfophthalein; BSO, D,Lbuthionine-[S,RI-sulfoximine; DEM, diethyl maleate; TLC, thin layer chromatography; HPLC. high-pressure liquid chromatography; v/v/v, volume to volume to volume; w/v, weight to volume.