The genetic difference in the susceptibility of mice to environmental toxicities induced by dioxin and related chemicals is governed by polymorphism of the arylhydrocarbon receptor (AhR) (Poland, A., and Knutson, C. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 517-554). cDNA cloning of AhR cDNA from responder (C57BL/6) and non-responder (DBA/2J) mice allowed us to analyze the structure and function of these AhRs. Both of the AhRs, which were expressed in COS-7 cells transfected with their expression plasmids, showed a clear 9 S complex with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) in a linear glycerol gradient centrifugation, consistent with the result of the endogenously expressed AhR in Hepa-1 cells. This result provides the first direct evidence that the cDNA-encoded protein binds the ligand specifically. Scatchard plot analysis revealed that the dissociation constant (Kd) of C57BL AhR for TCDD is 0.27 nM, while that of DBA AhR is elevated up to six times that high. Chimeric plasmids between the two cDNAs and site-directed mutagenesis revealed two critical alterations responsible for the reduced ligand binding activity: an Ala375 to Val alteration and an elongated carboxyl-terminal sequence due to a T to C mutation at the first letter of the termination codon of C57BL AhR. Two variants with reduced and intermediate ligand binding activity were also found in human AhRs with amino acid alterations equivalent to those of the DBA AhR. Importance of the amino acid at position 381 of human AhR (equivalent to position 375 of mouse AhR) in the ligand binding was confirmed by the fact that the mutation of Val381 to Asp completely abolished the ligand binding activity of human AhR.