Polycyclic aromatic hydrocarbons (PAHs) are a major source of air, water, and soil pollution. The multidrug resistance (mdr)/permeability glycoprotein (P-gp) complex is implicated in the multidrug resistance pattern developed against various drugs and xenobiotics, including polycyclic aromatic hydrocarbons. In order to develop a genomic biomarker, we investigated the response of the mdr49 gene (mdr49) of Drosophila melanogaster to PAHs. Structural analysis of mdr49-PA, which is the putative protein expressed from Drosophila mdr49 gene, demonstrated that this transmembrane protein indeed belongs to the adenosine triphosphate-binding cassette transporter superfamily. Polymerase chain reaction (PCR) and real-time PCR analysis revealed that the mdr49 gene is expressed continuously at all the stages of fly development, including embryos, pupae, larvae, and adults, as well as in embryonic Drosophila S12 cells. In the adult fly, the mdr49 gene was expressed in all the analyzed segments (head, thorax, and abdomen) and organs (olfactory and sexual organs). The quantification of mdr49 transcripts by real-time PCR in adult flies exposed to benzo[a]pyrene over time or in presence of increasing concentrations of this pollutant showed a clear dose-dependent response. Similarly, mdr49 gene expression increased after adult flies were exposed to structurally varied PAHs. The detection of tested PAHs by Drosophila P-gp efflux pump was checked by flow cytometry.