Hepatocyte nuclear factor-4 (HNF-4) is a liver-enriched transcription factor that is crucial in the regulation of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism and in determining the hepatic phenotype. We have previously shown that HNF-4 contains transcription activation functions at the N terminus (AF-1) and the C terminus (AF-2) which work synergistically to confer full HNF-4 activity. Here, we show that HNF-4 recruits the CREB-binding protein (CBP) coactivator on promoters of genes that contain functional HNF-4 sites. HNF-4 interacts with the N-terminal region of CBP (amino acids 1-771) and the C-terminal region of CBP (amino acids 1812-2441). The two activating functions of HNF-4, AF-1 and AF-2, interact with the N terminus and the N and C terminus of CBP, respectively. In addition, we show that in contrast to the other nuclear hormone receptors the interaction between HNF-4 and CBP is ligand-independent. Recruitment of CBP by HNF-4 results in an enhancement of the transcriptional activity of the latter. CBP does not activate gene expression in the absence of HNF-4, and dominant negative forms of HNF-4 prevent transcriptional activation by CBP, suggesting that the mere recruitment of CBP by HNF-4 is not sufficient for enhancement of gene expression. These findings demonstrate that CBP acts as a transcriptional coactivator for HNF-4 and provide new insights into the regulatory function of HNF-4.