Retinoic acid receptors, RARs, are retinoic acid (RA)-inducible transcriptional regulatory proteins which transduce the RA signal at the level of gene expression via a retinoic acid response element. Three subtypes of RARs have been described termed RAR-alpha, RAR-beta, and RAR-gamma. RARs, like other members of the steroid/thyroid hormone receptor family, are composed of six structurally distinct domains, one of which is responsible for binding RA. No structural information is available concerning the nature of the amino acids which are responsible for binding of RA within the ligand binding domain of any RAR. In this report, the role of 2 positively charged amino acids of RAR-beta for binding of RA, Arg269 and Lys220, was examined using site-directed mutagenesis. When compared with wild type RAR-beta, mutation of either Arg269 or Lys220 singly to the small neutral amino acid Ala had only a small effect on both the EC50 value in all-trans-RA and 9-cis-RA transactivation assays and the apparent Kd for all-trans-RA. However, mutation of both of these positively charged amino acids simultaneously to Ala caused a 500- and 100-fold elevation in the EC50 for all-trans-RA and 9-cis-RA, respectively, compared with that of wild type RAR-beta. Similarly, the apparent Kd for all-trans-RA was increased 580-fold when that of the double mutant was compared with that of the wild type RAR-beta. Furthermore, this double mutant RAR-beta acted as a dominant negative mutant when transfected with wild type RAR-alpha, -beta, or -gamma in a RA concentration-dependent fashion. Taken together these data demonstrate the importance of both Arg269 and Lys220 of RAR-beta for the binding of RA, possibly by interacting with the negatively charged carboxyl group of RA.