Recently, we identified a 315-bp intestinal enhancer (IE), localized over 55 kb upstream from the transcriptional start of the human apolipoprotein B (apoB) gene, that confers expression of human apoB transgenes in the intestines of mice. Four functional binding sites for the intestine-enriched transcription factors hepatocyte nuclear factor (HNF)-3beta, CAAT enhancer binding protein (C/EBP)beta, and HNF-4 were demonstrated within the 315-bp IE. In this report, we extend these earlier studies and examine the relative contributions of these three transcription factors to the activity of the enhancer as well as their mechanism of interaction with one another. Cotransfection experiments with the expression vectors for HNF-3beta, C/EBPbeta, and HNF-4 revealed that HNF-3beta bound to Site 1, C/EBPbeta bound to Site 2, and HNF-4 bound to Site 3 within the 315-bp IE and that the sites act synergistically to enhance intestinal expression of apoB. Each one of these four binding sites was mutated, and mutant constructs were transfected into intestine-derived CaCo-2 cells to evaluate the role of each of these binding sites in enhancer activity. The results of the mutagenesis experiments confirmed that the HNF-3beta and HNF-4 sites are most important for the enhancer activity, followed by C/EBPbeta Site 2. All three factors bound to Sites 1, 2, and 3 must act synergistically for optimal activity of the apoB IE.