Transcription of the glycoprotein hormone α gene is repressed by the thyroid hormone receptor (TR) in a hormone dependent manner. Previous studies identified a TR binding site immediately downstream of the TATA box. Site directed mutagenesis and transient gene expression studies were used to evaluate the role of this TR binding site as a negative thyroid response element (nTRE). Mutagenesis of the putative negative thyroid response element (nTRE) site eliminated TR binding but failed to eliminate negative regulation by T3. A mutation which converted the putative nTRE to a higher affinity palindromic element did not enhance repression, but rather eliminated thyroid hormone dependent negative regulation. Proximal α promoter sequences between −100 and + 44 were replaced with a heterologous thymidine kinase promoter resulting in a construct that was not repressed by T3 treatment. This finding confirmed that repression required proximal α promoter sequences and also indicated that repression did not occur by interference with the function of upstream the α gene enhancers. These studies indicate that TR binding adjacent to the TATA box is not required for T3 mediated repression of the α promoter and suggest that negative regulation may involve protein-protein interactions with promoter-specific transcription factors.