Cytochrome P450 (P450) enzymes encoded have generally been described as detoxifying enzymes. However, study of the insect steroid hormone 20-hydroxyecdysone (20E) revealed a group of CYP genes named Halloween Genes that participate in biosynthesis of the 20E precursor ecdysone, as well as conversion of ecdysone to the active hormone 20E. 20E is the primary regulator of insect developmental transitions, triggering molting and metamorphosis upon binding to the nuclear receptor heterodimer EcR-USP. In the fruit fly Drosophila melanogaster, there are 18 identified nuclear receptor-encoding genes presenting all 6 of the main nuclear receptor subfamilies in humans, while 20E is the only known active steroid-derived ligand. In addition to 20E, some ecdysteroids are present in insect hemolymph only at specific developmental stages, suggesting that there might be some unknown ecdysteroids serving as ligands for nuclear receptors other than EcR. Phylogenetic analysis showed a group of P450 genes evolutionarily close to Halloween Genes with unidentified physiological functions. Therefore, we propose to investigate the function of these uncharacterized P450 genes. The results of this thesis work suggest that one of the uncharacterized P450 genes, cyp303a1, has important functions in Drosophila development during pupal stage. Moreover, further study also indicates that cyp303a1 expression is required for activation of the dHNF4 nuclear receptor signaling pathway.