The chromatin architecture of a promoter is an important determinant of its transcriptional response. For most target genes, thyroid hormone receptor (TR) activates gene expression in response to thyroid hormone (T3). In contrast, the TSHa gene promoter is downregulated by TR in the presence of T3. Here we utilize the capacity for the Xenopus oocyte to chromatinize exogenous nuclear-injected DNA to analyze the chromatin architecture of the TSHa promoter and how this changes upon TR-mediated regulation. Interestingly, in the oocyte the TSHa promoter is positively regulated by T3. In the inactive state the promoter contains six loosely positioned nucleosomes. The addition of TR/RXR by itself has no effect on the chromatin structure but the inclusion of T3 induces strong positioning of a dinucleosome in the proximal TSHa promoter that is bordered by regions that are hypersensitive to cleavage by methidiumpropyl EDTA. We identify a novel thyroid response element that coincides with the proximal hypersensitive region. Furthermore, we examine the consequences of mutations in TR that impair coactivator recruitment. In a comparison with the xTRbA promoter, we find that the effects of these mutations on transactivation and chromatin remodeling are significantly more severe on the TSHa promoter.