Chemical pollution including endocrine disruptors is serious environmental problem for human and wildlife. Although screening systems of estrogenic endocrine disruptors have been established, available system of ecdysone-disrupting compounds for arthropod has not been established yet.   Here we developed a novel screening system of the endocrine disruptors for arthropod using mammalian cells. A human embryonic kidney cell line (HEK293 cells) stably expressing both Drosophia ecdysone receptor (EcR) and mammalian retinoid X receptor (RXR) was transfected with a plasmid possessing ecdysone response elements and reporter gene lacZ to generate EcR-293/lacZ cells. The EcR-293/lacZ cells were used for screening of ecdysone-disrupting compounds. Brief principle of this screening system is as follows: ecdysone-disrupting compound is initially bound to EcR/RXR heterodimer, these complexes of ligand and receptor are subsequently bound to ecdysone response elements and induce the transcription of lacZ gene, and β-galactosidase of EcR-293/lacZ cells is finally produced. Using this reporter gene assay system, we demonstrated that a phytoecdysone polypodine B induced β-galactosidase activity as well as a phytoecdysone ponasterone A and a synthetic ecdysteroid muristerone A. However, ecdysone, 2-deoxy-20-hydroxyecdysone, and 20-hydroxyecdysone, which are biologically active and endogenous ecdysteroid hormones for insects and crustacean, could not induce the β-galactosidase activity. In addition, ponasterone A inducing β-galactosidase activity was partially inhibited by 20-hydroxyecdysone. Furthermore, all-trans-retinoic acid, which is a ligand for RXR, could not affect the reporter gene assay. These results suggest that our developed EcR293/lacZ cells are suitable for screening of environmental phytoecdysteroids which possess ability to be bound to their own EcR/RXR receptor complex.