Liver-specific gene expression is controlled by a heterogeneous group of hepatocyte-enriched transcription factors. One of these, the winged helix transcription factor hepatocyte nuclear factor 3beta (HNF3beta or Foxa2) is essential for multiple stages of embryonic development. Recently, HNF3beta has been shown to be an important regulator of other hepatocyte-enriched transcription factors as well as the expression of liver-specific structural genes. We have addressed the role of HNF3beta in maintenance of the hepatocyte phenotype by inactivation of HNF3beta in the liver. Remarkably, adult mice lacking HNF3beta expression specifically in hepatocytes are viable, with histologically normal livers and normal liver function. Moreover, analysis of >8,000 mRNAs by array hybridization revealed that lack of HNF3beta affects the expression of only very few genes. Based on earlier work it appears that HNF3beta plays a critical role in early liver development; however, our studies demonstrate that HNF3beta is not required for maintenance of the adult hepatocyte or for normal liver function. This is the first example of such functional dichotomy for a tissue specification transcription factor.