The ligand binding domains (LBDs) from the EcR and USP proteins of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were purified as recombinant heterodimers. The K(d) values for [(3)H]-ponasterone A binding by LBD heterodimers that included the hinge regions (i.e., DE/F heterodimers) ranged 0.7-2.5 nM, with K(i) values for ecdysteroid and dibenzoylhydrazine ligands ranging from 0.1 nM to >448 microM. The K(d) and K(i) values for a recombinant H. armigera LBD heterodimer that lacked D-regions (i.e., an E/F heterodimer) were approximately 4 times higher than those for its DE/F counterpart. Rate constants were estimated for the L. cuprina LBD heterodimer. A fluorescein-inokosterone conjugate (K(i)~40 nM) was used to develop a novel binding assay based on fluorescence polarization. This assay, which ranked the affinity of competitor ecdysteroids in the same order as the [(3)H]-ponasterone A binding assay, is well suited to high-throughput screening. Ponasterone A had a higher affinity than muristerone A for the recombinant hemipteran LBD heterodimers, whereas the reverse was true for the recombinant dipteran one. The same preference was observed when these ligands were tested as inducers of ecdysone receptor-controlled gene expression in transfected mammalian cells. The binding data obtained in vitro using recombinant LBD heterodimers reflects the ability of agonists to induce transgene expression in recombinant mammalian cells, and can also reflect their efficacy as larvicides.