N-tert-Butyl-N,N'-dibenzoylhydrazine and its analogs are agonists of insect molting hormone (20-hydroxyecdysone: 20E). In order to elucidate the mode of action of these nonsteroidal ecdysone agonists at the molecular level, we expressed the receptor protein for 20E - the heterodimer of ecdysone receptor (EcR) and ultraspiracle (USP) - in vitro, and analyzed their binding affinities to ligands. For the lepidopteran Chilo suppressalis, we showed quantitatively that the receptor-binding activity of non-steroidal ecdysone agonists determines the strength of their molting hormonal and insecticidal activities. The binding activity of five representative non-steroidal ecdysone agonists to C. suppressalis EcR/USP was higher than that to dipteran Drosophila melanogaster ECR/USP, which probably results in the selective toxicity between these two insects.