We have validated a method to assess hepatic glutathione turnover in individual animals in vivo. This method would be applicable to man by collection of bile samples via nasoduodenal intubation. The rate of glutathione turnover was calculated from the time course of the specific activity of the glutathione-acetaminophen adduct in bile after the administration of a radiolabeled glutathione precursor and a small dose of acetaminophen. Identical results were obtained with radiolabeled glutathione or with radiolabeled cysteine, glutamic acid or glycine as the precursors. The small dose of acetaminophen administered to trap glutathione as an excretable adduct did not stimulate glutathione turnover, which reflects glutathione synthesis under steady-state conditions. No evidence for two pools of glutathione with different half-lives was found; previous reports of two glutathione pools may have failed to account for hepatic protein turnover with subsequent release of radiolabeled amino acids for glutathione synthesis. In male rats, the rate of glutathione turnover decreased from 0.52 per hr at 6 weeks of age to 0.12 per hr at 24 weeks of age. After acute depletion of glutathione by diethylmaleate, the rate of glutathione turnover promptly doubled in all age groups. Similar increases in the rate of glutathione synthesis and in the ability to stimulate glutathione production in response to acute depletion in children might explain their decreased susceptibility to acetaminophen hepatotoxicity.