Hormonal responsiveness in peripheral tissues is variable in patients with resistance to thyroid hormone (RTH). One cause of this may be differential interaction of RTH mutants of thyroid hormone receptor beta (TR beta) with TR auxiliary proteins (TRAPs). We used gel shift mobility assays to examine the interaction of wild-type and mutant TR beta s with retinoid X receptors (RXRs) and endogenous TRAPs. Some mutants showed reduced homodimerization but retained heterodimerization with recombinant RXRs. Wild-type TR beta formed heterodimeric complexes with multiple TRAPs in nuclear extracts of rat tissues, but RTH mutants showed variably altered heterodimerization with each TRAP. With liver nuclear extract, all mutants with impaired homodimerization also showed impaired TR beta-TRAP heterodimerization. Thus heterodimerizations with RXRs and TRAPs are differently affected by RTH mutations. Our results suggest that multiple TRAPs are expressed in tissue-specific patterns. The variability of TR beta heterodimerization with TRAPs may account, in part, for the variable tissue responsiveness in RTH.