The aryl hydrocarbon receptor (AhR) is a ligand-dependent, basic helix-loop-helix Per-Arnt-Sim (PAS)-containing transcription factor that can bind and be activated by structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Our previous three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain allowed identification of the binding site and its experimental validation. We have extended this analysis by conducting comparative structural modeling studies of the ligand binding domains of six additional highaffinity mammalian AhRs. These results, coupled with site-directed mutagenesis and AhR functional analysis, have allowed detection of the "TCDD binding-fingerprint" of conserved residues within the ligand binding cavity necessary for high-affinity TCDD binding and TCDD-dependent AhR transformation DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD with both wild-type and mutant mAhRs. Taken together, our results dramatically improve our understanding of the molecular determinants of TCDD binding and provide a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure. © 2009 American Chemical Society.