ABSTRACT: BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify novel HNF4alpha target genes in the human intestinal epithelial cells in order to elucidate the role of HNF4alpha in the intestinal differentiation progress. METHODS: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4alpha binding to promoter regions. The HNF4alpha ChIP-chip data was matched with gene expression and histone H3 acetylation status of the promoters in order to identify HNF4alpha binding to actively transcribed genes with an open chromatin structure. RESULTS: 1,541 genes were identified as potential HNF4alpha targets, many of which have not previously been described as being regulated by HNF4alpha. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH), and the tight junction protein cingulin (CGN) promoters verified that these genes are bound by HNF4alpha in Caco2 cells and for the Cdx-2 and trehalase promoters the HNF4alpha binding was verified in mouse small intestine epithelium. CONCLUSION: The HNF4alpha regulation of the Cdx-2 promoter unravels a transcription factor network also including HNF1alpha, all of which are transcription factors involved in intestinal development and gene expression.