The aryl hydrocarbon receptor (AHR) binds many aromatic hydrocarbon compounds and mediates their carcinogenesis. We demonstrate that the endogenous AHR physically associates with the endogenous TRAP/DRIP/ARC/Mediator complex in a ligand-dependent manner. The Med220 subunit, which is known to interact with several nuclear hormone receptors through its LXXLL motifs, potentiates AHR-dependent reporter gene activity in an LXXLL-independent manner. Depletion of Med220 substantially reduces endogenous AHR-mediated transcription from the mouse cytochrome P4501A1 gene (CYP1A1). Both Med220 and CDK8 (another subunit of TRAP/DRIP/ARC/Mediator) are recruited to the CYP1A1 enhancer in a TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-dependent fashion in vivo, and Med220 LXXLL motifs are not required. Med220 rapidly and persistently associates with the enhancer but not the promoter of the CYP1A1 gene after TCDD treatment with similar kinetics as AHR and the coactivators p300 and p/CIP. Our findings demonstrate a novel role for Med220 in AHR-regulated transcription that differs mechanistically from its role in transcriptional regulation by other previously studied transcription factors.