Chronic exposure to phenobarbital (PB) in the drinking water of male B6C3F1 mice starting at 4 weeks of age and subsequent to a single (ip) injection of diethylnitrosamine (DENA) administered on Day 15 of age has been shown to result in the inhibition of hepatic tumor formation. In this study, we varied the time of onset of PB administration to determine if sexual maturity would affect liver tumor formation and progression. Male B6C3F1 mice were divided into eight groups. Groups 1-4 received a single (ip) dose of 5 mg/kg DENA at 15 days of age while mice in groups 5-8 received saline. At weaning (4 weeks of age), groups 1 and 5 received deionized drinking water (DDW) for 24 weeks; groups 2 and 6 received PB (500 ppm) in the drinking water (PB DW) for 16 weeks followed by DDW for 8 weeks; groups 3 and 7 received DDW for 4 weeks, PB DW for 16 weeks, and then DDW for 4 weeks; and groups 4 and 8 received DDW for 8 weeks and PB DW for 16 weeks. Mice were killed at 28 weeks of age and hepatic lesions were evaluated. Mice which did not receive DENA (groups 5-8) exhibited no liver tumors. Animals in groups 1-4 exhibited hepatocellular foci and adenomas. PB treatment in groups 2, 3, or 4 resulted in a significant decrease in the incidence of DENA-initiated hepatocellular foci and adenomas when compared to those observed in group 1. The number of foci in group 4 was significantly decreased compared to those in groups 2 and 3. There was no significant difference in the adenoma incidence among groups 2, 3, and 4. No significant differences were observed in the sizes of foci or adenomas among groups 1-4. Data from this study suggest that the inhibition of hepatocellular tumorigenesis by PB remains intact even when the start of the administration of PB is withheld up to 12 weeks of age.