||Pulses of the steroid hormone ecdysone induce changes in gene expression that lead to the onset of metamorphosis in Drosophila melanogaster. The large number of nuclear receptor superfamily members that are regulated by ecdysone, as well as the functional interactions among vertebrate members of this superfamily, suggest that combinations of nuclear receptors may dictate tissue- and stage-specific responses to ecdysone. To understand how nuclear receptors may function together during the onset of metamorphosis, all of the superfamily members expressed at this stage must first be identified. As a step toward this goal, an extensive molecular screen was conducted to identify new members of the nuclear receptor superfamily that are expressed during the onset of Drosophila metamorphosis. Three novel genes identified in this manner, DHR38, DHR78, and DHR96, are true members of the nuclear receptor superfamily. DHR38 is the Drosophila homolog of the vertebrate NGFI-B receptor. Analysis of the DNA-binding specificities of DHR78 and DHR96 reveals that these proteins bind to distinct subsets of ecdysone receptor binding sites in vitro. This suggests that these receptors may function together with the ecdysone receptor at the top of the ecdysone-induced regulatory hierarchies. To test this hypothesis, a genetic analysis of DHR78 was conducted. Loss-of-function mutations in DHR78 cause lethality during the third larval instar, prior to the onset of metamorphosis. Genes normally induced by ecdysone during the third larval instar show little or no expression in DHR78 mutants. Localization of DHR78 protein on salivary gland polytene chromosomes demonstrated that this protein binds to a subset of ecdysone receptor binding sites in vivo, including most of the known ecdysone-regulated puff loci. In addition, the lack of phenotypes from ectopic expression of DHR78 suggests that this receptor is regulated posttranslationally. Together, these data suggest that DHR78 is a novel ligand-activated receptor that functions as a critical regulator of ecdysone responses during Drosophila development.