The aryl hydrocarbon receptor (AhR) is involved in various processes such as cytochrome P450 (P450) 1A induction after xenobiotic exposure. It is also considered to play a major role in cell proliferation and differentiation. Recent evidences have suggested a cross-talk between AhR functions and the mitogen-activated protein kinase (MAPK) cascade. We now report that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a specific inhibitor of MAPK kinase (MEK) MEK1/2, elicits a marked increase in CYP1A1 expression at both mRNA and protein levels associated with a significant increase of enzyme activity in primary rat hepatocytes and a human hepatoma cell line. This induction occurred independently of MEK/extracellular signal-regulated kinase (ERK) activation and in the absence of ERK1 and ERK2 expression. The effect of U0126 was mediated by its ability to transactivate xenobiotic responsive element (XRE)-driven genes, as demonstrated by transfection assays with an XRE-driven luciferase construct in the human B16A2 hepatoma cell line. CYP1A1 modulation was abolished by a cotreatment with resveratrol, an established AhR antagonist, arguing for AhR activation by U0126. Such an effect was demonstrated by direct in vitro ligand binding competition assays using rabbit liver cytosol, showing that this compound binds AhR with an EC 50 ϭ 25 ϫ 10 Ϫ6 M. Moreover, we demonstrated that U0126 is a substrate for several P450s including human CYP1A2, -1A1, and -1B1. We conclude that the widely used specific inhibitor of MEK/ ERK, U0126, also acts as a potent AhR activator and an inducer of related genes. Such effects on the AhR may have an impact on biological functions attributed previously to MAPK inhibition.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAH) are potent inducers of several genes, including some encoding "Phase I" and "Phase II" xenobiotic-metabolizing enzymes. These enzymes include cytochrome P450 (P450), glutathione transferases, NADPH: quinone reductases, and UDP-glucuronosyl transferases.TCDD and PAH effects are mediated by activation of the aryl hydrocarbon receptor (AhR), a cytosolic protein that forms complexes with two 90-kDa heat shock proteins and some other proteins. After ligand binding, the AhR is translocated and localized in the nucleus followed by an heterodimerization with the AhR nuclear translocator (Arnt) protein and acts as a transcriptional factor. Several reports have also shown constitutive activation of the AhR in the absence of exogenous ligand under certain conditions (Singh et al