Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta and gamma) regulate transcription of the transthyretin (TTR) and numerous other liver-specific genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of developmental regulatory proteins including the Drosophila homeotic fork head (fkh) protein. The mammalian HNF-3/fkh family consists of at least thirty distinct members and is expressed in a variety of different cellular lineages. In addition to the winged helix motif, several HNF-3/fkh family members also share homology within transcriptional activation region II and III sequences. In the present study we further define the sequence boundaries of the HNF-3 beta N-terminal transcriptional activation domain to extend from amino acids 14 to 93 and include conserved region IV and V sequences. We also demonstrate that activity of the HNF-3 N-terminal domain was diminished by mutations which altered a putative alpha-helical structure located between amino acid residues 14 and 19. However, transcriptional activity was not affected by mutations which eliminated two conserved casein kinase I sites or increased the number of acidic amino acid residues in the N-terminal domain. Furthermore, we determined that the nuclear localization signal overlaps with the winged helix DNA-binding motif. These results suggest that conserved sequences within the winged helix motif of the HNF-3/fkh family may be involved not only in DNA recognition, but also in nuclear targeting.