Alpha-albumin (AFM), a member of the albumin gene family that also includes albumin, alpha-fetoprotein, and vitamin D-binding protein, is expressed predominantly in the liver and activated at birth. Here, we identify two hepatocyte nuclear factor 1 (HNF1)-binding sites in the AFM promoter that are highly conserved in different mammals. These two sites bind HNF1α and HNF1β. The distal site (centered at -132, relative to AFM exon 1) is more important than proximal site (centered at -58), based on HNF1 binding and mutational analysis in transfected cells. Our data indicate that HNF1α is a more potent activator of AFM promoter than is HNF1β. However, HNF1β can act in a dominant manner to inhibit HNF1α-dependent transactivation of the AFM promoter when both proteins are expressed together. This suggests that the differential timing with which the albumin family genes are activated in the liver may be influenced by their responsiveness to HNF1α and HNF1β. Our comparison of HNF1-binding sites in the promoters in the albumin family of genes indicates that the primordial albumin-like gene contained two HNF1 sites; one of these sites was lost from the albumin promoter, but both sites still are present in other members of this gene family.