We previously generated a transgenic mouse line (T-77) in which increased hepatic expression of the hepatocyte nuclear factor-3beta (HNF-3beta) protein was used to assess its role in hepatocyte-specific gene transcription. The T-77 transgenic mice displayed elevated serum bile acid and bilirubin levels and a complete absence of hepatic glycogen storage. These postnatal liver defects were associated with diminished expression of hepatocyte genes involved in gluconeogenesis and bile acid transport as well as reduced levels of hepatocyte transcription factors. In this study, we show that mouse tail vein injections of adenovirus expressing the rat HNF-3beta (AdHNF3beta) cDNA efficiently increased its levels throughout the liver lobule and recapitulated the T-77 transgenic liver phenotype within several days postinfection. Likewise, the AdHNF3beta-infected liver phenotype was associated with reduced hepatic expression of genes involved in glucose homeostasis, bile acid transport, and bilirubin conjugation, which were not found with control adenovirus infections. These studies show that adenovirus-mediated gene transfer is an effective method for rapid hepatic increases in transcription factor levels to determine in vivo target genes. In contrast, AdHNF3alpha-infected liver displayed only a transient reduction in hepatic glycogen levels and was associated with less severe decreases in hepatic expression of gluconeogenic and bilirubin metabolism genes. Consistent with these findings, only T-77 transgenic and AdHNF3beta-infected liver exhibited diminished hepatic expression of the HNF-6 transcription factor, suggesting that reduced HNF-6 levels contribute to diminished HNF-3beta-specific transcriptional activity.