Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-induced signals stimulated by genotoxic agents. The present study is the first to investigate the mechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuring the effects of methylating agents on MAPK activity, DNA methylation, and intracellular glutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters were compared in a fetal rat lung cell line model (MP48). These compounds were chosen because they methylate DNA via a methanediazonium intermediate and, therefore, should induce similar cellular methylation patterns, although they produce different side products upon decomposition. All three compounds stimulated the activation of the stress-activated MAPKs, c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylating agents, these compounds also stimulated the activation of extracellular signal regulated kinase (ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widely considered to be the critical toxic lesion induced by methylating agents, including AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appear to be a key regulator of MAPK activity by these compounds, however. There is no direct relationship between the levels of O6-mG and the levels of MAPK activation, and formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. The present studies also indicate that depletion of glutathione is not required or sufficient to stimulate MAPK activation by the methylating agents investigated here. The use of a pharmacological inhibitor indicates that these methylating agents activate ERK through a signaling pathway that requires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylating agents activate MAPKs through mechanisms that are likely to involve the alkylation of cellular targets other than DNA.