We recently reported that an 8-kilobase (kb) region, spanning from -54 to -62 kb 5' of the human apolipoprotein B (apoB) gene, contains intestine-specific regulatory elements that control apoB expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal control region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity studies uncovered a prominent DNaseI hypersensitivity site, located within a 315-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcriptionally active CaCo-2 cells but not in transcriptionally inactive HeLa cells. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and analysis of the DNA sequence revealed putative binding sites for the tissue-specific transcription factors hepatocyte nuclear factor 3beta, hepatocyte nuclear factor 4, and CAAT enhancer-binding protein beta. Binding of these factors to the 315-bp enhancer was demonstrated in gel retardation experiments. Transfection of deletion mutants of the 315-bp enhancer revealed the relative contributions of these transcription factors in the activity of the apoB intestinal enhancer. The corresponding segment of the mouse apoB gene (located -40 to -83 kb 5' of the structural gene) exhibited a high degree of sequence conservation in the binding sites for the key transcriptional activators and also exhibited enhancer activity in transient transfection assays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp enhancer conferred intestinal expression to human apoB transgenes.