The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo 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 vivo, and during liver perfusions with BSP-containing perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo 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 vivo 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 novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-Km for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3μmol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.