Phenobarbital (PB), a liver-tumor promoter, at a concentration of 3 mM dramatically inhibited the growth of adult rat hepatocytes in the chemically defined medium, HGM, with added hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In concurrence with these findings, PB down-regulated expression of the HGF receptor (c-met) and suppressed production of the autocrine growth factor transforming growth factor-α (TGF-α). Furthermore, PB down-regulated expression of transcription factors associated with proliferation such as AP1 and NF-κB. In the presence of PB, hepatocytes remained morphologically differentiated and restoration of the expression of mature hepatocyte markers, such as albumin and cytochrome P450s (1A, 2B1/2, and 2E1), was accelerated after an initial phase of growth. Additionally, PB strongly suppressed expression of the mRNA for α-fetoprotein, a protein primarily expressed by fetal liver, and the accelerative effect of PB on restoration of mature hepatocyte markers showed a correlation with the up-regulation of the hepatocyte-enriched transcription factors HNF3 and HNF4. When the effects of PB on various extracellular matrix proteins were examined, the data indicated that PB specifically suppressed laminin and fibronectin production by hepatocytes, suggesting an important role for these proteins in growing hepatocyte cultures.