Isolated hepathocytes lost about half their content of GSH when treated in vitro with 90 nmol of diethylmaleate per 10(6) cells. Within one hour GSH started to accumulate in the cells, and the rate of accumulation was taken as a measure of the GSH synthesis rate. The rate was affected by additions of amino acids and horse serum to the incubation medium. The methionine and cysteine uptake rates were much lower than the rate of GSH synthesis and not affected by variations in the GSH synthesis rate. The methionine and cysteine uptake rates were not affected by horse serum. It is concluded that even though exogenous sulphur-containing amino acids facilitate GSH synthesis, the hepatic cysteine pool is to a large extent replenished by endogenous amino acids derived from protein degradation. In particular, this is the case when the turnover rate of the cysteine pool is increased by drug metabolism.