The effect on hepatic glutathione (GSH) turnover of transpeptidation of substrate amino acids by gamma-glutamyl transferase (GGT) was evaluated in intact rats. The infusion of 5 mmol kg-1 of L-methionine, L-cysteine and glycylglycine promptly stimulated hepatic GSH turnover as measured by acetaminophen probe analysis. This stimulation was abolished by pretreatment of the animals with inhibitors of GGT. Glycine and phenylalanine, two amino acids that are poor substrates for transpeptidation by GGT, had no effect on GSH turnover. A methionine load led to a decrease in hepatic GSH from 6.40 +/- 0.6 to 4.1 +/- 0.4 mumol g-1 and significantly increased the plasma clearance of GSH from 4.76 +/- 0.09 to 6.05 +/- 0.36 ml min-1 100g bw-1. In the in situ perfused rat liver increasing concentrations of methionine in the perfusate stimulated the efflux of GSH from 19.5 nmol min-1 g liver-1 without added methionine to 34.1 and 56.3 nmol min-1 g liver-1 at 0.1 and 1.0 mM methionine, respectively. We conclude that the administration of amino acids or dipeptides that are good substrates for transpeptidation by GGT increases hepatic turnover of GSH. The stimulation of sinusoidal efflux of GSH by methionine indicates that an increased efflux is responsible for the increased rate of turnover of hepatic GSH. The associated increase in the plasma clearance of GSH suggests that by increasing the efflux of GSH the liver may provide more GSH for the extracellular transpeptidation of substrate amino acids.