In the bacterium Bacillus subtilis, the DNA-binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144-amino acid-residue carboxy terminal dimerization/ ligand-binding domain of the BmrR, named the BRC (BmrR C-terminus). Tetragonal crystals of ligand-free BRC take the space group P41212, or its enantiomorph P43212, with unit cell dimensions a = b = 76.3 Å, c = 96.0 Å, α = β = γ = 90°. Diffraction is observed to at least 2.7 Å resolution at room temperature. In addition, we determined the secondary structure content of ligand-free and rhodamine-bound BRC by circular dichroism. In the ligand-free form, BRC has considerable β-sheet content (41%) and little α-helix structure (13%). After BRC binds rhodamine, its β-sheet content increases to 47% while the α-helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.