We identified a quinolone resistance locus, flqB, linked to transposon insertion omega 1108 and fus on the SmaI D fragment of the Staphylococcus aureus NCTC 8325 chromosome, the same fragment that contains the norA gene. S. aureus norA cloned from flqB and flqB+ strains in Escherichia coli differed only in a single nucleotide in the putative promoter region. There was no detectable change in the number of copies of norA on the chromosomes of flqB strains, but they had increased levels of norA transcripts. Cloned norA produced resistance to norfloxacin and other hydrophilic quinolones and reduced norfloxacin accumulation in intact cells that was energy dependent, suggesting active drug efflux as the mechanism of resistance. Drug efflux was studied by measurement of norfloxacin uptake into everted inner membrane vesicles prepared from norA-containing E. coli cells. Vesicles exhibited norfloxacin uptake after the addition of lactate or NADH, and this uptake was abolished by carbonyl cyanide m-chlorophenylhydrazone and nigericin but not valinomycin, indicating that it was linked to the pH gradient across the cell membrane. Norfloxacin uptake into vesicles was also saturable, with an apparent Km of 6 microM, a concentration between those that inhibit the growth of flqB and flqB+ S. aureus cells, indicating that drug uptake is mediated by a carrier with a high apparent affinity for norfloxacin. Ciprofloxacin and ofloxacin competitively inhibited norfloxacin uptake into vesicles. Reserpine, which inhibits the multidrug efflux mediated by the bmr gene of bacillus subtilis, which is similar to norA, abolished norfloxacin uptake into vesicles as well as the norfloxacin resistance of an flqB mutant, suggesting a potential means for circumventing quinolone resistance as a result of drug efflux in S. aureus. These findings indicate that the chromosomal flqB resistance locus is associated with increased levels of expression of norA and strongly suggest that the NorA protein itself functions as a drug transporter that is coupled to the proton gradient across the cell membrane.