The QacR multidrug-binding repressor protein regulates the expression of the Staphylococcus aureus qacA gene, a multidrug resistance (MDR) locus that is prevalent in clinical isolates of this important human pathogen. In this paper we demonstrate that the range of structurally diverse compounds capable of inducing qacA transcription is significantly more varied than previously appreciated, particularly in relation to bivalent cations. For all of the newly identified inducing compounds, induction of qacAexpression was correlated with a matching ability to dissociate QacR from operator DNA. Development of a ligand-binding assay based on intrinsic tryptophan fluorescence permitted dissociation constants to be determined for the majority of known QacR ligands, with values ranging from 0.2 to 82 microM. High-affinity binding of a compound to QacR in vitro was not found to correlate very strongly with either its in vivo inducing abilities or its structure. The latter observation indicated that the QacR ligand-binding pocket appears to have evolved to accommodate a wide range of toxic hydrophobic cations, rather than a specific class of compound. Importantly, the antimicrobial ligands of QacR included several plant alkaloids that share structural similarities with synthetic MDR substrates. This is consistent with the suggestion that the qacA-qacR MDR locus was recently derived from genes that protect against natural antimicrobial compounds.