A set of thirty-two natural and ten semisynthetic ecdysteroids was assayed in murine 3T3 cells across ten different ecdysteroid receptor (EcR) ligand-binding domains derived from nine arthropod species in an engineered gene switch format. Among the ecdysteroids tested, the most biologically wide-spread ecdysteroid, 20-hydroxyecdysone (20E), was moderately and consis-tently potent across the tested EcRs.