Abstract Administration of the nonsteroidal ecdysteroid agonist RH-5849 or 20-hydroxyecdysone to final larval instar tobacco hornworm, Manduca sexta, has been shown to induce an ecdysteroid inactivation enzyme, ecdysteroid 26-hydroxylase, and the cytosolic inactivation enzymes ecdysone oxidase and ecdysteroid phosphotransferase. In this work, we show that induction of ecdysteroid 26-hydroxylase by the ecdysteroid agonists RH-5849, RH-5992 (tebufenozide), and RH-0345 (halofenozide) appears universal in lepidopteran species that show susceptibility to the agonists. Interestingly, the waxmoth, Galleria mellonella, which shows very low susceptibility to the agonists but whose ecdysteroid receptor is capable of binding the agonist, shows no induction of a 26-hydroxylase activity. It appears that the more potent ecdysteroid agonists in Lepidoptera, RH-5992 and RH-0345, show in general a greater induction of 26-hydroxylase than RH-5849. Feeding RH-5849 to the dipteran housefly Musca domestica results in the induction of an ecdysteroid phosphotransferase. The low toxicity of these ecdysteroid agonists in orthopteran and coleopteran orders also correlates with a lack of induction of ecdysteroid 26-hydroxylase activity. We propose that in species where ecdysteroid agonists are effective in stimulating an untimely premature molt, a response to a state of hyperecdysonism elicited by the agonists is induction of enzymes of ecdysteroid inactivation.