Similar to certain unliganded steroid hormone receptor complexes, the unliganded aryl hydrocarbon receptor has been shown to consist of a multimeric core complex that includes the 90-kDa heat shock protein (hsp90) and the immunophilin-like hepatitis B X-associated protein 2 (XAP2). Immunophilins and XAP2 associated with these complexes bind to the carboxyl-terminal end of hsp90 through an interaction with their tetratricopeptide repeat (TPR) domains. The consensus TPR binding motif contains two domains, A and B. Recently, the carboxyl terminus of XAP2 has been shown to contain a highly conserved TPR domain that is required for the assembly of XAP2 with both hsp90 and AhR. A search of the murine AhR sequence identified domain B (A-F-A-P) of the consensus TPR sequence directly adjacent to the carboxyl-terminal side of the helix-loop-helix region of the murine and human AhR. We hypothesized that this conserved domain B region may be involved with mediating interactions between either AhR-hsp90, AhR-XAP2, and/or AhR-AhR nuclear translocator protein. Site-directed mutagenesis of the amino-terminal alanine residue of this region to an aspartic acid (A78D) completely inhibited 2,3,7, 8-tetrachloro-p-dioxin (TCDD) -dependent activation of a xenobiotic response element (XRE) driven gene expression construct in transfected COS-1 and BP8 cells. The A82F mutation caused a 40 to 50% decrease in TCDD-dependent activation. The inability of A78D and the reduction of A82F to trans-activate XRE-driven reporter activity did not result from impaired AhR-XAP2-hsp90 interactions, TCDD-dependent AhR translocation to the nucleus, or AhR-AhR nuclear translocator protein interactions. In vitro DNA binding analysis demonstrated that loss of trans-activation potential by the A78D mutation resulted from impaired XRE binding. This study underscores the potential importance of AhR mutations that occur naturally outside of known functional domains.