The staphylococcal multidrug resistance gene, qacA, confers resistance to an exceptional number of antimicrobial agents, many of which are commonly used as antiseptics and disinfectants. Importantly, the plasmid-borne qacA gene is carried by prevalent clinical isolates of the human pathogen Staphylococcus aureus. The QacA 514-amino acid exporter possesses 14 transmembrane alpha-helices (TMS) and belongs to the major facilitator superfamily (MFS) of transport proteins. It mediates resistance to structurally-unrelated antimicrobials via a proton motive force-dependent mechanism. To gain further insight into this mechanism QacA amino acid residues Ser106 through to Pro139, proposed to be situated within TMS4 and the loops between TMS3:TMS4:TMS5, were subjected to cysteine-scanning mutagenesis. Thirty four residues were individually substituted with cysteine and resulting QacA protein derivatives examined for a number of characteristics including; the ability to efflux the substrate ethidium, solvent accessibility of introduced cysteines as gauged by fluorescein maleimide binding, as well as determination of the resistance profile to a representative set of six antimicrobial compounds. A number of residues were identified, that when substituted, showed a reduced ability to efflux ethidium and/or altered resistance to selected antimicrobials. The majority of these functionally-important residues are highly conserved in the drug/proton antiporter (DHA) family of MFS drug export proteins. Fluorescein maleimide was able to strongly label a number of residues within TMS4 and preincubation with maleimide reduced ethidium efflux for some mutants. Taken together, these results identify a number of residues in the TMS 4 region of QacA essential for the binding and export of cationic antimicrobial compounds and confirm the topology model of QacA predicted by hydropathy analysis.