Transcriptional coactivators, CREB-binding protein (CBP) and p300, exhibit high homology in structure and similar functions. In the present study, we analyzed the function of CBP and p300 proteins as transcriptional coactivators in the expression of albumin in hepatocytes. The expression levels of CBP and p300 were high in fetal hepatocytes, but low in adult ones. Immunoprecipitation assays showed that both CBP and p300 interacted with hepatocyte nuclear factor-1alpha (HNF-1alpha) in primary hepatocytes. Furthermore, CBP and p300 were co-precipitated without HNF-1alpha. Chromatin immunoprecipitation (ChIP) assays revealed that both CBP and p300 are located in the albumin promoter region in hepatocytes. These results suggested that HNF-1alpha, CBP and p300 were incorporated into a preinitiation complex of RNA polymerase II at the albumin promoter. Luciferase reporter assays showed that CBP and p300 cooperatively triggered HNF-1alpha-mediated transcription of the albumin promoter. In addition, inhibition of CBP or p300 using small interfering RNAs (siRNAs) resulted in a reduction in albumin expression. These results suggest that both CBP and p300 are required for enhanced expression of albumin.