Complementary DNAs for two mutant thyroid hormone α1 receptors (TRα1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TRα1 (w-TRα1), which bound these TREe with different homodimer/monomer ratios, binding of mutant L to the three TREe as homodimers was reduced by ~90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by ~80%. Surprisingly, its binding to Pal was markedly enhanced compared with w- TRα1. The binding of these two mutants to the three TREe as heterodimers with retinoid X receptors (RXRα and -β) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DP4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TRα gene de occur in patients and that these novel TRα1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.