Resistance to the juvenile hormone analog, methoprene, in Drosophila melanogaster has been identified as a target site mutation in a bHLH-PAS protein similar to the mammalian aryl hydrocarbon receptor (AhR), which regulates cytochrome P450 genes in vertebrates. To determine if cytochrome P450 genes are regulated by methoprene and/or the methoprene protein (Met), transcriptional expression of cytochrome P450 subfamilies and genes was compared between developmental stages, methoprene-sensitive (Oregon-R) and tolerant (Rst(1)JH¹, Met²⁷) strains of Drosophila melanogaster. In third instars of Oregon-R, methoprene in the diet was associated with decreased expression of Cyp6g1 and Cyp4g15 while the other P450s did not respond to methoprene. Expression of Cyp6d5, Cyp6d4, Cyp314a1 and Cyp4e2 was higher and expression of Cyp4d1 and Cyp4g15 was lower in the two methoprene tolerant strains. Methoprene modulated expression of P450s differently in the two strains. Constitutive expression of Cyp6a2 was negligible in Rst(1)JH¹ but present in Oregon-R. A deletion in the promoter of Cyp6a2 from Rst(1)JH¹ may be responsible for the lack of expression. The results demonstrate that mutating the Met protein changes the expression of cytochrome P450 genes in D. melanogaster, while methoprene modulates that expression in a Met-dependent manner. Enrichment of the promoters with putative response elements may explain differences in the response to methoprene and the Met receptor. Correlating methoprene-regulated expression with putative response elements will help in our understanding of the molecular mechanisms of both insecticide resistance and metamorphosis in insects.