NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli, whereas the converse is true of enzymes from Staphylococcus aureus. This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.