Phenobarbital (PB), a non-genotoxic hepatocarcinogen in rodents, has been studied extensively but its mechanism of carcinogenic action is unclear. PB appears to function as a tumor promoter by selectively inducing the growth of preneoplastic hepatocytes. In the present study, the comparative effects of PB at tumor-promoting and non-promoting doses were examined in male B6C3F1 mice and male F344 rats. In addition, the mechanism by which PB produced the selective induction of preneoplastic cell growth (increased DNA synthesis/cell proliferation and/or decreased apoptosis) was investigated. Preneoplastic focal lesions were produced using diethylnitrosamine (DEN). After the lesions were histologically apparent, mice and rats were fed PB (10, 100, or 500 mg/kg NIH-07 diet) or control diet and sampled after 7, 30 and 60 days of treatment In both mice and rats, 100 and 500 mg PB/kg increased the number and the relative volume of focal lesions. In rats and mice, 10 mg PB/kg did not enhance focal lesion growth. The preneoplastic lesions that clonally expanded due to phenobarbital treatment were predominantly eosinophilic in appearance. In addition, DNA synthesis in focal hepatocytes was significantly increased in the 100 and 500 mg PB/kg diet. In PB-treated mice and rats, there also was a significant decrease in the rates of apoptosis in focal hepatocytes. Therefore, our data showed that PB at doses of 100 and 500 mg/kg diet promoted focal hepatic lesion growth both by increasing DNA synthesis and cell proliferation and by decreasing the rate of apoptosis.