Thyrotropin releasing hormone (TRH) gene is regulated negatively at the transcriptional level by thyroid hormone (T3) in rat anterior hypothalamus. The actions of T3 upon other target genes are known to be mediated through the thyroid hormone receptors (TR), TR alpha and TR beta. To explore whether the inhibitory regulation of human (h) TRH gene transcription by T3 is TR isoform specific and whether TRH gene transcription can be modulated as well by unliganded TR isoforms, transient gene expression studies have been carried out using hTRH-luciferase (TRH-Luc) chimeric constructs and TR expression constructs, co-transfected into a human neuroblastoma cell line (HTB-11). Data herein demonstrate T3-dependent inhibitory regulation of the hTRH gene promoter by TR-T3 complexes. Moreover, significant inhibition (39%-60%) could be achieved by T3 bound to either hTR alpha 1, hTR beta 1, or rTR beta 1, beta 2 and was comparable quantitatively, indicating an absence of TR isoform specificity for T3 inhibition. Conversely, basal promoter activity of the hTRH gene could be activated significantly by unliganded hTR alpha 1, beta 1, rTR beta 1, and beta 2 (150% to 334%), but not by hTR alpha 2. Thus, TRs appear to exert opposite effects on hTRH gene transcription, depending on the presence or absence of ligand (T3). These dual effects of TR suggest that the addition of the T3 ligand effects conformational changes that can abrogate the initiation of transcription.