The characteristics and kinetics of glutathione (GSH) efflux were examined in homogeneous suspensions of freshly isolated rat hepatocytes. GSH efflux was measured as its linear accumulation in the suspension medium. Appearance of GSH extracellularly was reflected in a quantitative loss in cellular GSH. However, the total GSH remained essentially unchanged, indicating minimal net synthesis of GSH under these experimental conditions. GSH efflux was sensitive to temperature, with a calculated Q10 value of 2.3. A wide range of cellular GSH concentration ranging from near complete and moderate depletion to severalfold the control values was achieved by treatment of animals or cells with various GSH depletors or inducers. At physiological (fed) and elevated (3-methylcholanthrene- and CoCl2-induced) cellular GSH, the rate of GSH efflux was near maximum. The rate fell dramatically to 50% maximum at a GSH concentration equaling 35 nmol/10(6) cells. A 48-h fast resulted in a 40% loss of cellular GSH, with a corresponding decrease in efflux rates. Addition of GSH to the incubation medium had no effect on efflux rates. The relationship of GSH efflux to cellular GSH concentration was characterized by apparent sigmoidal saturation kinetics. The data were fitted well by the Hill model with the following kinetic parameters: Vmax = 0.25 nmol X 10(6) cells-1 X min-1, Km = 3.5 mM, and n = 3. These results correspond very closely to our previous findings in the perfused liver.