The mechanisms by which the methylating agent methyl methanesulfonate (MMS) kills cultured hepatocytes were studied. In an amino-acid-free Krebs-Ringer buffer (KRB), 1 mM MMS depleted the cells of glutathione (GSH) within 1-2 hr. Lipid peroxidation, as measured by the accumulation of malondialdehyde (MDA), followed, and over 70% of the cells died within 3-4 hr. The iron chelator deferoxamine and the antioxidant N,N'-diphenyl-1,4-phenylenediamine (DPPD) prevented lipid peroxidation and death of the hepatocytes without any effect on the loss of GSH. 3-Aminobenzamide (ABA), a poly(ADP-ribose) polymerase inhibitor, also prevented the cell killing by attenuating the loss of GSH. In a culture medium containing amino acids and antioxidants (Williams' E medium, WEM), 1 mM MMS killed the hepatocytes more slowly, with 70% of the cells dying 8-12 hr after treatment. Lipid peroxidation accompanied the loss of viability. Deferoxamine and DPPD inhibited lipid peroxidation, while only partially protecting against the cell killing. ABA offered more protection and reduced the decline of GSH and decreased lipid peroxidation. ABA also reduced the extent of the depletion of both NAD and ATP that accompanied the cell killing by MMS in WEM. These data indicate that MMS killed the hepatocytes by different mechanisms depending on the culture conditions. In KRB, the toxicity of MMS was a consequence of oxidative cell injury that follows the depletion of GSH. In WEM, both oxidative injury and the action of poly(ADP-ribose) polymerase in response to DNA single-strand breaks contributed to the loss of viability.