Hepatic glutathione turnover and the efflux of glutathione from the liver into bile and blood were measured in male Sprague-Dawley rats in vivo. In fed rats the efflux of glutathione into blood, calculated from the hepatic arteriovenous concentration gradient and hepatic blood flow, amounted to 12.4 ± 1.4 nmoles liver. Together with the excretion of glutathione into bile (3.4 ± 0.4 nmoles per liver) total efflux accounted for the hepatic turnover of glutathione of 15.2 ± 0.9 nmoles per liver. Fasting animals for 48 hr markedly increased hepatic glutathione turnover to 26.4 ±1.2 nmoles per min-gm liver. Increased efflux into blood rather than increased intrahepatic catabolism accounted for this increased turnover. The systemic clearance of glutathione was 3.22 ± 0.51 ml per min 100 gm body weight. The efflux of glutathione from liver therefore was calculated to contribute over 90% of total glutathione inflow into the circulation, as determined from the clearance and the arterial concentration of glutathione. Thus, the liver is the major source of plasma glutathione, and turnover of hepatic glutathione in the basal state is accounted for almost entirely by efflux of glutathione from the liver. During fasting, the plasma clearance of exogenous glutathione increased to 5.32 ± 0.35 ml per min-100 gm body weight, and the utilization of methionine for glutathione synthesis increased markedly. The increased extrahepatic catabolism during fasting results in a decrease in plasma glutathione, which in turn may account for the observed increase in sinusoidal glutathione efflux with concomitant stimulation of the rate of hepatic glutathione turnover and of synthesis. By utilizing methionine for glutathione synthesis, the liver thus is able to make more cysteine available to other organs when dietary sources of cysteine are deficient.