The intracellular glutathione redox state was estimated using newly adapted methods for tissue analysis. Under standard perfusion conditions of rat liver perfused in situ, intracellular GSH content was 5.5 mumol X g of liver-1, and intracellular GSSG content was 18 nmol X g of liver-1, resulting in a GSH/GSSG ratio of 300. GSSG was transported from the cells into bile. The control rate of release amounts to 0.4 nmol X min-1 X g of liver-1 and corresponds to the rate of release previously observed in anesthetized animals (Sies, H., Koch, O., Martino, E., and Boveris, A. (1979) FEBS Lett. 103, 287-290). No GSSG was released into the caval perfusate. In contrast, GSH release occurs both into bile and into the sinusoidal space, the rates of release being 1 and 14 nmol X min-1 X g of liver-1, respectively. Biliary GSH release in very low in isolated perfused liver. The relationship between intracellular GSSG levels and the rate of biliary GSSG transport was studied. This was achieved in experiments in which internal hydrogen peroxide formation was stimulated with substrates for monoamine oxidase (pargyline-sensitive), or by addition of t-butyl hydroperoxide, or of nitrofurantoin. The apparent concentration ratio, GSSG in bile/GSSG intracellular, was about 50 over the range of intracellular GSSG examined.