Negative regulation by thyroid hormone is mediated by nuclear thyroid hormone receptors (TRs) acting on thyroid hormone response elements (TREs). We examine here the role of human TR-β2, a TR isoform with central nervous system-restricted expression, in the regulation of target genes whose expression are decreased by triiodothyronine (T3). Using transient transfection studies, we found that TR-β2 achieved significantly greater ligand-independent activation on the thyrotropin-releasing hormone (TRH) and common glycoprotein α-subunit genes than either TR-β1 or TR-α1. A chimeric TR-β isoform containing the TR-β2 amino terminus linked to the TR-α1 DNA- and ligand-binding domains functioned like the TR-β2 isoform on these promoters, confirming that the amino terminus of TR-β2 was both necessary and sufficient to mediate this effect. By constructing deletion mutants of the TR-β2 amine terminus, we demonstrate that amino acids 89-116 mediate this function. This domain, important in ligand-independent activation on negative TREs, is discrete from a previously described activation domain in the amino-terminal portion of TR-β2. We conclude that the central nervous system-restricted TR-β2 isoform has a unique effect on negative regulation by T3 that can be mapped to amino acids 89-116 of the amino terminus of the human TR-β2.