Pulses of the steroid hormone 20-hydroxyecdysone (20E) trigger the larval-to-adult metamorphosis of Drosophila by reprogramming gene expression throughout the organism. 20E directly induces a small set of early regulatory genes that repress their own expression and induce a large set of late secondary-response genes. We show here that two members of the Drosophila nuclear hormone receptor superfamily, DHR3 and DHR39, are rapidly induced by 20E, in parallel with the early regulatory genes. Both genes also require protein synthesis at high 20E concentrations for their maximal induction by the hormone. Developmental Northern blot analysis reveals that DHR39 is induced in mid third instar larvae and expressed throughout most of third instar larval and prepupal development, while DHR3 is briefly expressed in late third instar larvae and early prepupae. The 20E-induction and temporal patterns of DHR3 and DHR39 transcription strongly suggest that these genes function together with the early regulatory genes to coordinate the complex gene networks that direct the early stages of Drosophila metamorphosis. In an initial effort to understand how these two orphan receptors might function during development, we examined their DNA binding properties and compared them with the known Drosophila nuclear receptor superfamily members that are involved in the ecdysteroid response: EcR, Usp, E75A, E78A, and beta FTZ-F1. Upon testing all pairwise combinations of these seven proteins on a panel of seven oligonucleotides, only EcR and Usp bound DNA as a heterodimer, indicating that this interaction is highly specific. With the exception of E78A, which did not bind any sequence tested, each of the remaining proteins is able to bind to a single consensus AGGTCA half-site; however, each displayed different specificities depending on the flanking nucleotide sequence. These observations suggest that the 20E-regulated orphan receptors function as monomers to control the expression of their target genes.