In this report we have characterized further the myelin basic protein (MBP) gene thyroid hormone response element (TRE) by functional and binding analysis. Mutation and deletion experiments revealed that this TRE, confined to the sequences -184 to -167 of the MBP promoter, is able to function as a classical regulatory element in the context of the native and a heterologous promoter. It is comprised of two regions, containing a motif that is highly conserved among other TREs: AGGACA, arranged as an inverted palindrome. Any mutation within the footprinted region impaired receptor binding and function. Moreover, the deletion of sequences outside of the receptor footprinted region (MBP-TRE-18) resulted in a higher triiodothyronine responsiveness and a concomitant increase in receptor-dependent, hormone-independent repression. Results of transfection assays showed that both receptors alpha and beta elicit indistinguishable triiodothyronine responses when the MBP-TRE functions as a regulator of a heterologous promoter activity. However, a preferential beta receptor transactivation was observed when the MBP-TRE was placed in the context of its native promoter.