Many nuclear hormone receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA as either homodimers or heterodimers with the 9-cis-retinoic acid receptor (RXR). Protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains contribute to this binding. We have previously reported that the ligands for VDR and RXR can modulate the affinity of the receptors' interaction with DNA [Cheskis, B., & Freedman, L. P. (1994) Mol. Cell. Biol. 14, 3329-3338]. To examine this in more detail, we report here the use of surface plasmon resonance (SPR) to characterize the kinetics of both protein-protein and protein-DNA interactions by VDR and RXR in the presence and absence of their cognate ligands. We find that 1,25 dihydroxyvitamin D3 binding favors both VDR-RXR heterodimerization and, as a result, DNA binding by the complex. Conversely, the ligand reduces VDR homodimerization in solution and the affinity of VDR-DNA interaction. 9-cis-Retinoic acid attenuates the stimulating effect of 1,25-dihydroxyvitamin D3 by decreasing the rate of VDR-RXR heterodimer formation and simultaneously by increasing the affinity of RXR homodimerization. Thus, using SPR, we have shown that a major role for such ligands is to regulate nuclear receptor dimerization both in solution and on DNA. The ligands appear to do so dynamically, modulating the overall affinity of these complexes. This mechanism therefore creates a fast and sensitive way to regulate DNA binding in response to changes in ligand concentration.