The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D₃ receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.