The specificity of expression in the liver of the human apolipoprotein (apo) E/C-I gene locus is determined by a hepatic control region (HCR) that is located 15 kilobases downstream of the apoE gene. DNase I footprint studies of this sequence using nuclear extracts identified a region of the HCR that is enriched in nuclear protein-binding sites. Nuclease analysis of chromatin revealed liver-specific DNase I-hypersensitive sites that were associated with this region, and additional liver-specific nuclease-sensitive sites associated with the apoE gene were identified. The HCR domain has a limited binding affinity for the nuclear scaffold. The specific domain required for liver expression was tested by ligating subfragments of the HCR to the apoE gene and examining their activity in transgenic mice. A segment of 319 nucleotides that contained several potential regulatory sequences was required for full activity of liver-specific transcription with shorter segments yielding much lower levels of expression in the liver. All constructs that contained a fully active HCR were expressed in approximately a copy-dependent manner, suggesting that transgene expression was independent of integration position. Taken together, the properties of the HCR are consistent with its function as a locus control region for the liver-specific expression of the apoE gene.