Phenobarbital (PB) is a non-genotoxic liver tumor promoter used extensively in initiation-promotion protocols. To determine the mode of PB action, double transgenic mice overexpressing both the c-myc and transforming growth factor (TGF)-alpha genes were treated with PB in the food for 10 weeks, from 3 weeks of age. After 3-4 weeks on PB a peak in liver mass was noted, which subsequently leveled off at a value approximately 30% above untreated animals. The mitotic index in mice given PB peaked at 1 week of treatment and was significantly elevated compared with untreated animals. No significant difference between treated and untreated animals was seen thereafter, although a trend of PB-associated mitotic suppression was noticeable. The apoptotic index also showed a trend of suppression compared with untreated animals, significant after prolonged PB administration. Dysplastic hepatocytes were more prominent in PB-treated mice than untreated animals, particularly pericentrally. Removal of PB from the diet at 4 weeks of treatment led to a dramatic increase in apoptosis. This accompanied a drop in the liver mass to the level of untreated controls by 10 days. Throughout the study, PB-treated animals showed markedly lower levels of TGF-beta1 ligand, coincident with an elevated level of the anti-apoptotic protein Bcl-2. On withdrawal of PB, the levels of all these proteins rapidly changed to mirror those seen in untreated mice. In all treatment groups, no change in the levels of epidermal growth factor receptor, TGF-beta receptors I and II or Bcl-xS/L were seen. We conclude from our data that PB stimulates liver growth in double transgenic c-myc/TGF-alpha mice by induction of liver hypertrophy and inhibition of apoptosis, brought about by both a decrease in signaling through the TGF-beta pathway and an increase in Bcl-2. The data support the hypothesis that PB promotes neoplastic development through a reduction in the incidence of cell death.