The effect of acetaminophen (APAP) exposure on the formation of oxidized glutathione (GSSG) was investigated in cultured mouse hepatocytes to determine if oxidative damage is involved in the toxicity of this drug. Incubations of hepatocytes for 24 hr with 1 mM APAP produced a time-dependent loss of cell viability which was preceded by depletion of reduced glutathione (GSH) and an increase in GSSG formation. Pretreatment with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) (0.1 mM) for 30 min, which irreversibly inhibited glutathione reductase (GSSG-Rd) activity, increased the extent of GSSG formation produced by APAP exposure and potentiated its cell killing. Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for 1 hr to chelate ferric iron decreased GSSG formation and cell killing produced by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation as determined by the formation of the APAP-GSH conjugate or the covalent binding of APAP metabolites to cellular protein. Hence, increasing the susceptibility of hepatocytes to an oxidative stress with BCNU increased both the formation of GSSG and cell killing produced by APAP. Conversely, decreasing their susceptibility to an oxidative stress by chelating iron with DFO decreased GSSG formation and cell injury. It follows that APAP toxicity involves oxidative processes that occur early in the poisoning process and are a major factor contributing to injury in these cells.