Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor α (TRα) from ligand-treated HeLa (α-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRα, and these interactions are mediated through the C terminus of TRα and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor α, retinoid X receptor α, peroxisome proliferation-activated receptor (PPAR) α, PPARγ and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor α, PPARα, and PPARγ. Consistent with these results, TRAP220 moderately stimulates human TRα-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRα) and in cell free transcription systems (TRα and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRα during the function of the TRα–TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.