The heterodimers of all three ecdysone receptor (EcR) isoforms with Ultraspiracle (Usp), the invertebrate orthologue of RXR, bind Ponasterone A with the same affinity in the absence of DNA. Ligand binding is stimulated by ecdysone response elements (EcREs) to different degrees depending on the receptor isoform, the heterodimerization partner, and the type of EcRE. Ligand binding to heterodimers with wtUsp is enhanced 5-fold with hsp27, Pal-1 and DR-1. In the presence of DNA substantial differences in ligand binding were observed, when the AB-domain of wtUsp is replaced by the N-terminus of VP16, which is routinely used for the determination of transcriptional activity to overcome the inhibitory action of the AB-domain of Usp. Enhanced dimerization in the presence of hormone response elements increases mainly the number of binding sites resulting in improved ligand binding, which is observed even if the C-domain of Usp is deleted. RXR, which can partially replace Usp function, confers high affinity ligand binding only in the presence of an EcRE.