Hepatocyte nuclear factor-1 (HNF-1) plays an important role in the regulation of a large number of genes expressed in the liver, kidney, and pancreatic beta-cells. In exploring the molecular mechanism involved in HNF-1-dependent gene activation in the in vivo chromatin context, we found that HNF-1 can physically interact with the histone acetyltransferases (HATs) CREB-binding protein (CBP), p300/CBP-associated factor (P/CAF), Src-1, and RAC3. The transcriptional activation potential of HNF-1 on a genome integrated promoter was strictly dependent on the synergistic action of CBP and P/CAF, which can independently interact with the N-terminal and C-terminal domain of HNF-1, respectively. Moreover, the HAT activity of both coactivators was important, as opposed to the selective requirement for the HAT activity of P/CAF in activation from a transiently transfected reporter. Interaction of CBP with the N-terminal domain of HNF-1 greatly increased the binding affinity for P/CAF with the C-terminal activation domain, which may represent the molecular basis for the observed functional synergism. The results support a model that involves the combined action of multiple coactivators recruited by HNF-1, which activate transcription by coupling nucleosome modification and recruitment of the general transcription machinery.