A 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase-like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV subunits and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP-dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature-sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co-expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single-point mutations occurring in the 'quinolone resistance-determining region' (QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser-83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone-resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low-quinolone-resistant isolates. In contrast, Ser-80 of GrlA, which corresponds to Ser-83 of E. coli GyrA, is substituted to Phe or Tyr in both high- and low-quinolone-resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.