The role of retinoid X receptors (RXRs) on negative thyroid hormone response elements (nTREs) is not well understood. In this report, we demonstrate that an orientation-specific monomeric thyroid hormone receptor (T3R) DNA-binding site mediates thyroid hormone inhibition in the thyrotropin beta subunit gene (TSH-beta) from human and murine species. Unlike positive TREs, addition of the ligand 9-cis retinoic acid (9-cis RA) to cells transfected with a T3R beta 1 expression vector significantly reduces thyroid hormone inhibition of the TSH-beta gene, indicating that endogenous retinoid receptors antagonize T3R function. Cotransfection of an RXR-alpha but not a retinoic acid receptor-alpha expression vector further antagonizes thyroid hormone inhibition, but only in the presence of 9-cis RA. Antagonism by RXR requires both an intact DNA- and ligand-binding domain. Removal of monomeric T3R binding to the TSH-beta nTRE also requires both RXR domains. A model is proposed whereby monomeric T3R is removed from a nTRE by RXR occupied by its ligand 9-cis RA. This is the first report of 9-cis RA-dependent antagonism of thyroid hormone inhibition via negative TREs.