The intact human growth hormone (hGH) gene is negatively regulated by triiodothyronine (T3) treatment in transfected rat pituitary tumor cells. We now demonstrate that this responsiveness is mediated by a negative thyroid hormone response element (nTRE) localized to the proximal 3'-untranslated/3'-flanking region (3'-UT/FR). This region binds thyroid hormone receptors specifically and with high affinity. nTRE function was promoter-dependent, since it suppressed the activity of a positive TRE in the human chorionic somatomammotropin promoter, partially repressed activity of the herpes simplex virus TK promoter, but did not function with the human actin or Rous sarcoma virus promoters. T3 treatment did not alter transcript termination sites nor selectively affect the stability of transcripts containing the hGH 3'-UT/FR when transcription was blocked by actinomycin D treatment. The function of the nTRE depended on its location in the 3'-UT/FR; it was inactive when positioned down-stream of the simian virus 40 (SV40) 3'-UT/FR, and it acted as a positive TRE when placed upstream of the hGH promoter. These results demonstrate a novel localization of a TRE with unique properties which suggests expanded mechanisms by which thyroid hormone receptors can affect gene expression.