Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor α (RXRα) in heart, DNA- protein binding studies were performed with an RXR-responsive element (NRRE- 1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRα and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1RXR·cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1- RXR·peroxisome proliferator-activated receptor, NRB-1-RXR·retinoic acid receptor, or NRRE-1RXR·thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE- 1RXR·CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRα and RXRγ but not with RXRβ, indicating a receptor subtype-specific binding preference and suggesting an RXR AB region-dependent interaction. The RXR·CERIP complex did not form on NRRE-1 when a mutant GST- RXRα fusion protein lacking the NH2-terminal AB region (but containing the receptor dimerization domain) of RXRα was added in place of the full-length RXRα, confirming a role for the AB region in the RXR·CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRα via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.