Previously, we have identified a hormone response element (gamma F-HRE) composed of an everted repeat of the half-site (A/G)GGTCA motif separated by 8 base pairs that mediates retinoic acid (RA) activation of the gamma F-crystallin promoter. Here, we report that this element is bound by the thyroid hormone (T3) receptor in the form of heterodimers with either the retinoid X receptor (RXR) or the retinoic acid receptor (RAR). The T3R/RXR heterodimer binds to this element with high affinity but the transcriptional activity of the T3 receptor on this element is effectively antagonized by RAR alpha. Thus, RAR alpha exerts a dominant effect on the gamma F-HRE-everted repeat by mediating both RA activation and preventing T3 response. Although RAR/T3R heterodimers bind to the gamma F-HRE, they do not appear to be involved in transcriptional regulation since they bind with low affinity, and their ability to bind DNA is dramatically decreased by T3. Repression requires the DNA- and ligand-binding domains of RAR alpha and is consistent with a competitive DNA binding model of repression. However, in vitro binding studies indicate that RAR/RXR heterodimers form less stable interactions with the gamma F-HRE compared with T3R/RXR heterodimers; this suggests that in vivo the binding affinity of RAR/RXR heterodimers may be enhanced by accessory factors.