Acetamide is a hepatocarcinogen in rats. We previously revealed that acetamide induces characteristic large micronuclei in rat liver, suggesting the possible involvement of chromosome aberrations in acetamide-induced hepatocarcinogenesis. To elucidate the mechanism of large micronuclei formation, in this study we examined time-dependent changes in rat hepatocytes after administration of acetamide. Male 6-week-old F344 rats were gavaged with a single-dose administration of acetamide. A liver micronucleus test showed large micronuclei formation 48 and 72 h after acetamide administration. Histopathological analysis showed binucleated hepatocytes with a unilateral atrophic nucleus beginning 6 h after acetamide administration, and the number reached a maximum at 24 h. At 48 h, the number of binucleated hepatocytes with an atrophic nucleus decreased, and apoptotic hepatocytes and large micronucleated hepatocytes appeared. The changes in the frequency of these abnormal binucleated hepatocytes demonstrated a transition from atrophic nuclei to large micronuclei. Immunohistopathological examinations of binucleated hepatocytes showed loss of nuclear lamina, accumulation of barrier-to-autointegration factor (BAF) and chromatin condensation with heterochromatinization at the atrophic site of nuclei. Results of a BrdU-labeling assay were negative. The abnormal expression of BAF in morphologically normal nuclei suggested that nuclear envelope aberration in hepatocytes was an initial event of the nuclear atrophy. In addition, lack of involvement of cell division in the nuclear atrophy and large micronucleus formation was also demonstrated by BrdU-labeling assay. Overall, our data suggest that large micronuclei induced by acetamide are formed in binucleated hepatocytes through nuclear atrophy.