Thyroid hormone (T3) exerts its many biological activities through interaction with specific nuclear receptors (TRs) that function as ligand-dependent transcription factors at genes that contain a thyroid hormone response element (TRE). Mutant TRs have been detected in human hepatocellular carcinoma cell lines and tissue, but their contribution to carcinogenesis has remained unclear. The interaction of four such mutant TRs (J7-TRα1, J7-TRβ1, H-TRα1, and L-TRα1) with transcriptional coregulators has now been investigated. With the exception of J7-TRα1, which in the absence of T3 exhibited transcriptional silencing activity with a TRE-reporter gene construct in transfected cells, the mutant TRs had little effect (compared with that of wild-type receptors) on transcriptional activity of the reporter gene in the absence or presence of T3, of the transcriptional corepressors SMRT, NCoR or of the transcriptional coactivator SRC. Electrophoretic mobility-shift assays revealed that, in the presence of T3, the J7-TRβ1 mutant did not interact with SRC, whereas J7-TRα1 and H-TRα1 exhibited reduced abilities to associate with this coactivator and L-TRα1 showed an ability to interact with SRC similar to that of wild-type TRα1. The dominant negative activity of the mutant TRs in transfected cells appeared inversely related to the ability of the receptors to interact with SRC. Whereas J7-TRβ1, H-TRα1, and L-TRα1 did not interact with SMRT, and NCoR. J7-TRα1 bind to corepressors but failed to dissociate from them in the presence of T3. These aberrant interactions between the mutant TRs and transcriptional coregulators may contribute to the highly variable clinical characteristics of human hepatocellular carcinoma.