ultraspiracle gene product(USP) is one of several orphan receptors in Drosophila, sharing significant homology with the mammalian retinoid X receptor. In Drosophila the response to the hormone is mediate in pat by USP and ecdysone receptor(ECR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcRE) and ecdysone to modulate transcription. We used the yeast two-hybrid assay for detection of protein-protein interactions in vivo to screen for novel partners of USP. The GAL4DNA-binding domain fused to USP was used as bait to screen a Drosophila embryonic cDNA library in which the cDNA was fused to the GAL4 activation domain. Several cDNA clones encoding proteins that interact with USP were isolated, one of which corresponded to the ecdysone receptor A isoform (ECR-A). Domain analysis on USP revealed that the ligand binding domain is required for heterodimerization with ECR-A. Given the ability of USP to dimerize preferentially with ECR-A, this strategy should be useful for cloning novel partners for USP from a variety of cell types.