To investigate the pathogenesis of hepatotoxicity by methylating agents, we exposed isolated hepatocytes to N-nitrosodimethylamine (NDMA), N-methyl-N'-nitro-N nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Although NDMA is a potent in vivo hepatotoxicant in rats, no evidence of hepatocyte injury, measured by the leakage of lactate dehydrogenase (LDH) activity into the medium, was observed following exposure to a 1-100 mM concentration of either NDMA or MNU. In contrast, exposure of hepatocytes to MMS or MNNG resulted in greater than or equal to 90% LDH release. These differences in toxicity were not related to the extent of covalent binding to hepatocytes. Following MMS or MNNG, but not MNU or NDMA exposure, a significant rise in the generation of thiobarbiturate (TBA)-reactive species was observed. When hepatocytes were exposed to the antioxidant promethazine prior to the addition of MMS or MNNG, the formation of TBA-reactive species was inhibited completely. Although promethazine blocked MNNG-mediated cell injury, the antioxidant had no effect on MMS intoxication. These data suggest that methylating agents can cause hepatotoxicity by more than a single mechanism. For MNNG, lipid peroxidation may be involved in the pathogenesis of acute hepatotoxicity.