We have developed an in vivo medium-term liver initiation assay system to detect initiation activities of chemicals on multi-organ carcinogenesis. However, cell proliferation stimuli during the test chemical treatment period, required in the previously used assay models using adult rats, are laborious; moreover, those cause decrease of hepatic metabolic enzymes and psychological and physical discomfort to animals resulting in inaccurate interpretation. Therefore, we investigated the utility of another in vivo medium-term liver initiation assay model using 4-week-old rats without the cell proliferation stimuli. In this study, we confirmed that 4-week-old and 4.5-week-old male rats have high hepatocyte proliferation activity and similar enzyme activities of hepatic Cytochrome P450 subtypes as compared with 8-week-old male rats. Next, the in vivo medium-term liver initiation assay model using 4-week-old rats without cell proliferation stimuli was evaluated for the detection of the initiation activity of 1,2-dimethylhydrazine (DMH), which is a well-known genotoxic carcinogen. Four-week-old rats were orally administered DMH (single dose, 4 or 16 mg/kg; or 4-day repeat, 1 or 4 mg/kg); subsequently, these rats were treated promotion treatment consisted of administration of 2-acetylaminofluorene and carbon tetrachloride. Four weeks after the first DMH administration, the glutathione S-transferase placental form (GST-P)-positive foci induced by DMH in the liver was measured immunohistochemically. The inductions of GST-P-positive foci in all DMH-treated groups were dose-dependent, duration-dependent and significantly higher than that in non-DMH-treated group. From these results, our assay model was detected the initiation activity of DMH simply, and would be useful to evaluate the carcinogenicity of chemicals.