Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4 alpha (HNF4 alpha) in the ABCG5/ABCG8 intergenic promoter, through which HNF4 alpha strongly activated the expression of a reporter gene in both directions. The HNF4ci-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4 alpha. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4 alpha leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4a and GATA were essential for maximal synergism. We also show that HNF4a, GATA4, and GATA6 colocallize in the nuclei of HepG2 cells and that a physical interaction between HNF4 alpha and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4 alpha acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.