Hepatocyte nuclear factor 1α (HNF1α) and HNF4α are liver-selective transcription factors and are essential for hepatocyte differentiation. This study demonstrates that HNF1α as well as HNF4α genes contain a direct repeat with a space of one nucleotide (DR1)-retinoic acid (RA) response element that can be bound and regulated by RA and retinoid x receptor α (RXRα) complex. Transient transfection experiments showed that RA increased the promoter activity of the HNF1α and HNF4α genes in Hep3B cells. Overexpression of RXRα further enhanced the activities of both genes. Two putative RXRα binding sites on the HNF1α (−295 to −276) and HNF4α (−418 to −399) genes have been characterized. By transient transfection, both sites positively responded to RA, and overexpression of RXRα in Hep3B cells increased the regulatory effect. Gel mobility shift assay demonstrated that these two DR-1 sites could be bound by RXRα specifically. These data suggest that the differentiation effect of RA on hepatocyte may be due to direct interaction of RXRα with the RA-responsive elements on the HNF1α and HNF4α genes.