Our working hypothesis states that DNA damage is a critical step in toxic cell death. The DNA hypothesis was tested in cultured mouse hepatocytes by examining whether inhibitors of DNA repair would increase dimethylnitrosamine toxicity and DNA damage in parallel. Inhibitors were chosen for selectivity toward DNA polymerase alpha (aphidicolin, myricetin), DNA ligase (ethidium bromide), or multiple repair enzymes (ara-C, doxorubicin). Dimethylnitrosamine caused concentration-dependent DNA damage at 6 hr and cell death at 24 hr (35% ALT release vs. 8.8% in control cultured hepatocytes). Each repair inhibitor increased dimethylnitrosamine-induced DNA damage and toxic cell death in parallel. Doxorubicin maximally elevated DNA fragmentation and toxicity (57% ALT release). Repair inhibitors alone failed to damage DNA or cause cell death in this model system. These data support the hypothesis that DNA damage is an early causal event in toxic cell death caused by alkylating hepatotoxicants.