The developmental events occurring during molting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the molting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20-hydroxyecdysone (20E), are mediated by four cytochrome P450 enzymes, encoded by genes in the Halloween family. Orthologs of the Drosophila Halloween genes phantom (phm: CYP306A1), disembodied (dib: CYP302A1), shadow (sad: CYP315A1) and shade (shd: CYP314A1) were obtained from the endocrinological model insect, the tobacco hornworm Manduca sexta. Expression of these genes was studied and compared with changes in ecdysteroid titer that controls transition from the larval to pupal stage. Phm, dib and sad (mediating the final hydroxylations in the biosynthesis of ecdysone; E) were selectively expressed in the prothoracic gland, the primary source of E during larval and pupal development. Changes in their expression correlate with the hemolymph ecdysteroid titer during the fifth (final) larval instar. Shd, the 20-hydroxylase, which converts E into the more active 20E, is expressed in tissues peripheral to the prothoracic glands during the fifth instar. Transcript levels of shd in the fat body and midgut closely parallel the enzyme activity measured in vitro. The data indicate that these Halloween genes are transcriptionally regulated to support the high biosynthetic activity that produces the cyclic ecdysteroid pulses triggering molting.