Previous studies have shown that naphthalene oxide and reactive naphthalene metabolites diffuse from intact, isolated hepatocytes. The amount of naphthalene oxide diffusing from the cells as a percentage of the total formed remained constant over a wide range of substrate concentrations, thus suggesting that depletion of glutathione might not be required prior to significant naphthalene oxide efflux. However, the relative intracellular versus extracellular covalent binding of reactive metabolites increased with increasing naphthalene concentrations, thereby suggesting that glutathione might be involved in modulating the extent of intracellular covalent binding. To examine this question in detail, intracellular glutathione levels were monitored in mouse hepatocytes incubated in the presence of various concentrations of naphthalene. Naphthalene produced a concentration- and time-dependent decrease in intracellular glutathione levels and, at higher concentrations, a marked decrease in the rate of glutathione efflux from hepatocytes. This decrease in hepatocellular glutathione levels correlated well with the shift in binding from predominantly extracellular to intracellular. Inclusion of glutathione and glutathione transferases in the cell incubation medium partially blocked the depletion of intracellular glutathione by naphthalene, thus suggesting that naphthalene oxide diffusing into the cell medium was partially responsible for intracellular glutathione depletion. Finally, in vivo administration of naphthalene oxide to mice produced a dose-dependent depletion of pulmonary but not hepatic or renal glutathione but only at doses that were greater than 75 mg/kg. These studies support the view that there is not a glutathione threshold for the efflux of naphthalene oxide from intact hepatocytes and suggest that naphthalene oxide is capable of diffusing into as well as out of isolated hepatocytes.