CCAAT displacement protein (CDP) is a transcriptional repressor that contains four distinct DNA-binding domains; a homeodomain and three cut repeats. Each DNA-binding domain of CDP was expressed as a glutathione S-transferase (GST)-fusion protein and analyzed for relative binding affinity to five CDP-binding sites within the gp91phox promoter. Each cut repeat exhibits a unique pattern of DNA-binding affinities for the five binding sites in the gp91phox promoter, suggesting that each may make a distinct contribution to the DNA-binding behavior of native CDP. Although measurement of DNA/protein complex mass indicates that an isolated cut repeat can bind DNA as a monomer, mixing of GST-cut repeat and GST-homeodomain fusion proteins enhances DNA-binding activity. Far-Western blot and two-hybrid analyses indicate, however, that the CDP domains do not directly interact. We hypothesize that GST-mediated dimerization leads to spatial juxtaposition of these DNA-binding domains, and that the resulting enhanced DNA-binding activity mimics cooperative interactions that occur between these domains in native CDP.