Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor that controls embryonal liver development and that maintains and regulates gene expression in adult liver cells. We have previously demonstrated that transient overexpression of HNF-4alpha up-regulates a number of liver-specific genes in hepatoma cell lines. In this study, we extend these studies by assessing the functional role of HNF-4alpha in regulating cellular viability and liver-specific functions of primary rat hepatocytes. In cells transfected with an adenovirus vector carrying rat HNF-4alpha cDNA, induction and maintenance of liver-specific genes and functions were observed over a long-term culture, which might be associated with the prevention of a rapid loss of the mitochondrial membrane potential. In addition, we demonstrated that transthyretin mRNA was up-regulated by HNF-4alpha in primary hepatocytes, but not in hepatoma cells. These results indicate that HNF-4alpha plays a role in the maintenance of morphologically and biochemically functional hepatocytes and that the difference in expression of liver-specific genes induced by HNF-4alpha may depend on a differentiation state of cells.