Abstract The interaction of N-(4-ethoxyphenyl) p-benzoquinone imine (NEPBQI), a metabolite formed during peroxidase catalyzed metabolism of p-phenetidine, with GSH and its effects in isolated rat hepatocytes were investigated. When reacted with GSH NEPBQI formed both a mono- and a diglutathione conjugate as well as GSSG. Formation of glutathione conjugates and GSSG also occurred when NEPBQI was added to isolated hepatocytes. The formation of GSSG was, however, only detectable if the hepatocytes had been pretreated with the GSSG reductase inhibitor BCNU (1,3-bis-(2-chloroethyl-1-nitrosourea). Similarly, NEPBQI caused a rapid decrease in cellular free protein thiols when added to hepatocytes, however this was expressed at higher concentrations than for effects on GSH. The protein thiol decrease was correlated with protein binding of NEPBQI. NEPBQI was also shown to be toxic to isolated hepatocytes. At a concentration of 400 μM extensive bleb formation was followed by loss of cell membrane integrity and cell death. To assess further the subcellular metabolism of NEPBQI microsomes and cytosol was used. NEPBQI was found to be preferentially reduced by cytochrome P-450 reductase in the microsomes whereas DT-diaphorase catalyzed its reduction in cytosol. NEPBQI did not undergo significant redox cycling since no formation of O 2̄ was observed. Thus, the cytotoxic effect of NEPBQI appears to be due to protein arylation rather than redox cycling.