The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 2 x 10(-5) to 1 x 10(-3).