The purpose of this study was to evaluate the impact of modulating the repair of O6-alkylguanine adducts on the developmental toxicity of alkylating agents. Alkylating agents that have been shown to induce developmental toxicity following either in vitro or in vivo exposure were chosen for this investigation, and include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). P19 cells are pluripotent murine embryonic carcinoma cells that can be induced by all trans retinoic acid (RA) to differentiate into cells that are biochemically and morphologically very similar to cells of the central nervous system. These cells are useful for studying the ability of chemicals to affect neuronal viability and differentiation. Neuronally differentiating P19 cells were pretreated with O6-benzylguanine (O6-Bg), a potent and specific inhibitor of the O6-alkylguanine-DNA-alkyltransferase (AT) protein that repairs lesions at the O6-position of guanine. In previous studies using micromass rat embryo midbrain cells, O6-Bg greatly potentiated the ability of MNU but not ENU to inhibit differentiation, and did not significantly alter the effects of either MNU or ENU on viability. In the P19 cells, we found that AT inhibition potentiated the effects of MMS, MNU, and EMS to inhibit both viability and differentiation. Additionally, AT inhibition had a much greater effect on toxicity of the methylating agents, as compared to the ethylating agents. These results suggest that O6-alkylguanine adducts can inhibit both viability and differentiation in P19 cells treated with alkylating agents.