Two norA genes associated with hydrophilic quinolone resistance in Staphylococcus aureus were identified on the two recombinant plasmids pMR8736 and pSA209; the former was derived from a quinolone-resistant strain MR8736, and the latter was derived from a fluoroquinolone-susceptible strain 209P. We compared functions of these two genes, norA8736 and norA209 respectively, by introducing them into E. coli MC1061. Both genes expressed a novel protein of 52 kilodalton (kD) in size in MC1061. However, only norA8736 could confer hydrophilic quinolone resistance to the host cell, which was accompanied by a significant decrease in the uptake of a hydrophilic quinolone, norfloxacin, by the cell. Subcloning and recombinant plasmid analyses localized the hydrophilic quinolone-resistance marker to the 0.5 kilobase (kb)-long HpaI-HinfI DNA fragment of pMR8736. Nucleotide sequencing of this region and the corresponding region of pSA209 revealed that the hydrophilic quinolone resistance conferred by norA8736 was caused by a single nucleotide substitution from A (adenosine) in norA209 to C (cytosine), which corresponded to a single amino acid substitution from Asp to Ala.