AbstractSudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 µM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20‐fold after 16 and 24 h. The transactivation was due to the activation of a region located at −1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III‐‐treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:16–22 2012; View this article online atwileyonlinelibrary.com. DOI 10.1002/jbt.20408