The multidrug efflux pump QacA from Staphylococcus aureus confers resistance to an extensive range of structurally dissimilar compounds. Fluorimetric analyses demonstrated that QacA confers resistance to the divalent cation 4',6-diamidino-2-phenylindole, utilizing a proton motive force-dependent efflux mechanism previously demonstrated for QacA-mediated resistance to the monovalent cation ethidium. Both the ionophores nigericin and valinomycin inhibited QacA-mediated export of ethidium, indicating an electrogenic drug/nH+ (n ≥ 2) antiport mechanism. The kinetic parameters, K(m) and V(max), were determined for QacA-mediated export of four fluorescent substrates, 4',6-diamidino-2-phenylindole, 3',3'-dipropyloxacarbocyanine, ethidium, and pyronin Y. Competition studies showed that QacA-mediated ethidium export is competitively inhibited by monovalent cations, e.g. benzalkonium, and non-competitively inhibited by divalent cations, e.g. propamidine, which suggests that monovalent and divalent cations bind at distinct sites on the QacA protein. The quaternary ammonium salt, 1-(4- trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, was used as a membrane- specific fluorescence probe and demonstrated that the amount of substrate entering the inner leaflet was significantly reduced in QacA-containing strains, supporting the notion that the substrate is extruded directly from the membrane.