Retinoid X receptors (RXRs) exert transcriptional activities through heterodimerization with members of the nuclear hormone receptor superfamily. RXRs also act as homodimers and stimulate transcription from an RXR responsive element (RXRE) when bound to 9-cis-retinoic acid (9cRA). Here direct effects of 9cRA have been examined on biochemical and mechanistic parameters of RXR beta. It is shown that 9cRA significantly increases RXR beta homodimer binding affinity to an RXRE (Kd without ligand = 18 nM, Kd with ligand = 6 nM), while decreasing significantly the affinity of RXR beta/thyroid hormone receptor (T3R alpha) heterodimer binding to the same element. Effects on other response elements are also examined. The RXR beta homodimer was found to contact both halves of the RXRE direct repeat, irrespective of the effect of added ligand, while the RXR beta/T3R alpha heterodimer contacted the element only through a specific half-site. Binding of the homodimer to the element functionally activates RXR beta, since RXR beta enhanced transcription in vitro from a specific template in a ligand-dependent fashion. In agreement, transfection of RXR beta alone (but not RXR beta/T3R alpha) led to ligand-dependent activation of a reporter containing the RXRE. Taken together, 9cRA facilitates functional activation of the RXR beta homodimer in an element-dependent manner.