Hepatocyte nuclear factor 4 (HNF-4) is an orphan intracellular receptor that appears to be a key factor in the regulation of energy metabolism. In order to gain greater understanding of the binding and activation requirements of HNF-4, we performed genetic analysis of the apoCIII promoter, a promoter that has previously been shown to be highly sensitive to HNF-4-induced transcription. We identified two elements within the apoCIII promoter that bind HNF-4, either of which are sufficient to confer promoter induction in response to HNF-4. These two elements are both direct repeat-like in nature, but they differ significantly in motif sequence and the repeats are separated by either 1 or 2 nt. Therefore, to better define the DNA sequence recognition requirements of HNF-4, we utilized PCR-based binding site selection. HNF-4 selected for direct repeat-like elements with either 1 or 2 nt between the repeats. Surprisingly, the strongest selection was for a core motif that included the nucleotides between the repeats. Mutation of the nucleotide between the repeats resulted in a 6-fold reduction in affinity, indicating that the interaction between HNF-4 and the intervening nucleotide(s) is critical for high affinity binding.