The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, and mdr3) encoding P-glycoprotein. We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene (3-MC). This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BPrc1 and the AhR nuclear translocator (Arnt)-defective BPrc1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR/Arnt. We show that the mdr1 promoter (-1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BPrc1 cells. Deletion analysis indicated that the region from -245 to -141 contains cis-acting sequences mediating the induction, including a potential p53 binding sequence. 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1 promoter in an AhR/Arnt-dependent manner. Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction. Benzo(a)pyrene (B(a)P), a PAH and AhR ligand which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR/Arnt, also activated p53 and induced mdr1 transcription. 2,3,7,8-tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect. These results indicate that the transcriptional induction of mdr1 by 3-MC and B(a)P is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation. The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1 independently of AhR/Arnt further supports the proposition that mdr1 is transcriptionally upregulated by p53 in response to DNA damage.