Treatment of the last-instar larvae of the cotton leafworm (Spodoptera littoralis) with ecdysteroids (moulting hormones) results in the induction of an ecdysteroid-inactivation pathway. Administration of ecdysone, 20-hydroxyecdysone or an ecdysteroid agonist, RH 5849, leads to induction of an ecdysteroid 26-hydroxylase activity. This induction occurred in both early sixth-instar larvae and in older larvae which had been headligated to prevent the normal developmental increase in ecdysone 20-mono-oxygenase activity. The induction of 26-hydroxylase activity requires both RNA and protein synthesis, as demonstrated by experiments involving actinomycin D and cycloheximide. The 26-aldehyde derivative of ecdysone and ecdyson-26-oic acid were also formed from ecdysone in the RH 5849-induced systems. Formation of the aldehyde and the corresponding 26-oic acid (ecdysonoic acid) from 26-hydroxyecdysone was directly demonstrated in a cell-free system, thus establishing the following inactivation pathway: