While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb) interacts with Hsp90 to control hormonal perception and signaling. Treatment with high Triiodothyronine (T3) concentrations induced higher Hsp90 expression in mouse fibroblasts. We can show by microarray-based displacement assay, micro-scale thermophoresis, streptavidin-conjugated quantum dot (SAv-QDs) masking assay or 8-anilino-1-naphthalene sulfone (ANS) fluorescence that T3 enhanced adenosine triphosphate (ATP) binding of Hsp90, while microarray-based interaction analyses revealed that Hsp90 bound directly TRb and was released by T3. The dose-dependent binding of Hsp90 to TRb revealed an affinity of 5 nM. The affinity near the physiological range suggests that the release of TRb induced by T3 additionally influences chaperone activity of Hsp90 when T3 increased over normal concentrations. The observation is that T3 interaction with TRb and Hsp90 is an amplifier of the cellular stress response.