The regulatory regions of the genes for coagulation Factors VIII and IX contain binding sites for both liver-enriched and ubiquitous transcriptional regulators. We investigated the role of the liver-enriched protein, hepatic leukemia factor (HLF), in mediating transcriptional regulation of the Factor VIII and IX genes. Using transient transfection assays in HepG2 hepatoma cells, we demonstrated the ability of HLF alone and in synergistic combination with the D-box binding protein (DBP), another proline and acidic-rich (PAR) protein family member, to transactivate these promoters. HLF is capable of binding to multiple sites in both the Factor VIII and Factor IX promoters. At least some of the synergistic activation of the Factor VIII promoter seen with HLF and DBP cotransfection can be attributed to increased binding of HLF-DBP heterodimers to two Factor VIII promoter sites. We have also demonstrated that an E2A-HLF chimera, derived from a t(17;19) translocation in pre-B acute lymphoblastic leukemia (ALL) cells, is capable of mediating expression from the Factor VIII and Factor IX promoters in both hepatoma cells and pre-B ALL cells. These observations indicate that the PAR family of transcription factors plays an important and complex role in regulating expression of the Factor VIII and Factor IX genes, involving the binding of both homodimeric and heterodimeric complexes of HLF and DBP to several sites in the promoters. Finally, these studies reaffirm the potential role of dimeric transcription factor complexes in mediating interactions with specific promoter elements, which, in the case of the Factor VIII promoter, results in dramatically enhanced binding of HLF-DBP heterodimers to two cis-acting sequences. These observations further our understanding of the role played by members of the PAR family of transcription factors in regulating expression of the Factor VIII and Factor IX genes.