Abstract The syndrome of resistance to thyroid hormone (RTH) encompasses a heterogenous group of conditions which are caused by mutations of thyroid hormone receptor β1 (TRβ1). Mutations usually cluster in two regions of the ligand-binding domain. The mutant receptors can inhibit normal receptor activity in a dominant negative manner, consistent with the dominant mode of inheritance of RTH. Recent evidence suggested that this dominant negative effect (DNE) of the RTH mutants involves competition for DNA binding and emphasized the essential role of intact DNA binding activity for mutants in order to exert DNE. However, we found that a Cys73Ser substitution in the DNA-binding domain (DBD) of wild-type produces a TR which can inhibit the transcriptional activation by TRα1, either in the presence or absence of T 3, on three different TRE-containing reporter genes, in transient co-transfection studies. Co-expression of TRvα2, a TRα splicing variant, can enhance this DNE. However, DNE was not observed on the negatively-regulated TSHα Luc reporter gene when wild-type and DBD mutant were co-transfected at equimolar ratios. The DNE of DBD mutant is not reversed by co-transfection with excess retinoid X receptor α. DBD mutant alone can also inhibit the transactivation from a TK-luciferase reporter gene either linked with rat malic enzyme thyroid response element, or not. These observations parallel those we previously observed using TRvα2. Our results indicate that a DBD mutant can have DNE, possibly through a mechanism similar to that of TRvα2, which may involve interference with basal transcription factors. The clinical significance of these DBD mutants is currently unclear, but it is logical to expect such mutants do occur in nature.