Previous studies have suggested that both cAMP-dependent signal transduction pathway and Ca2+/protein kinase C-dependent pathway are involved in GSH efflux from hepatocytes. In the present study, GSH efflux from Hep G2 cells, a human-derived hepatoma cell line, was further characterized. Both epidermal growth factor (0.1-10 ng/ml) and insulin (1 microgram/ml) significantly increased GSH efflux from Hep G2 cells. A fall in the membrane potential produced by the replacement of Na+ with equivalent K+ did not affect GSH efflux significantly. Neither ouabain, a Na+/K+ ATPase inhibitor, vanadate, a Ca2+ ATPase inhibitor, nor BaCl2, a K+ channel blocker, significantly affected the GSH efflux. Methionine (1mM) decreased GSH efflux from the cells, although total GSH content in the cells was not affected during the incubation time of 60 min. Signal transductions through tyrosine kinase-coupled receptors may also be involved in GSH efflux from hepatocytes.