Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called "Black Box", has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14 alpha-ol (Delta(4)-diketol) and 5 beta[H]cholesta-7-ene-3,6-dione-14 alpha-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Delta(4)-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Delta(4)-diketol or diketol. These results indicate that Delta(4)-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok. (C) 2012 Elsevier Inc. All rights reserved.