Thyroid hormone thyroxine (T₄) and tri-iodothyronine (T₃) production is regulated by feedback inhibition of thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) synthesis in the pituitary and hypothalamus when T₃ binds to thyroid hormone receptors (TRs) interacting with the promoters of the genes for the TSH subunit and TRH. All of the TR isoforms likely participate in the negative regulation of TSH production in vivo, but the identity of the specific TR isoforms that negatively regulate TRH production are less clear. To clarify the role of the TR-β2 isoform in the regulation of TRH gene expression in the hypothalamic paraventricular nucleus, we examined preprothyrotropin-releasing hormone (prepro-TRH) expression in mice lacking the TR-β2 isoform under basal conditions, after the induction of hypothyroidism with propylthiouracil, and in response to T₃ administration. Prepro-TRH expression was increased in hypothyroid wild-type mice and markedly suppressed after T₃ administration. In contrast, basal TRH expression was increased in TR-β2–null mice to levels seen in hypothyroid wild-type mice and did not change significantly in response to induction of hypothyroidism or T₃ treatment. However, the suppression of TRH mRNA expression in response to leptin reduction during fasting was preserved in TR-β2–null mice. Thus TR-β2 is the key TR isoform responsible for T₃-mediated negative-feedback regulation by hypophysiotropic TRH neurons.