In order to clarify the mechanism of action of quinolones against Staphylococcus aureus, GrlA and GrlB proteins of topoisomerase IV encoded by genes with or without mutations were purified separately as fusion proteins with maltose-binding protein in Escherichia coli. The reconstituted enzymes showed ATP-dependent decatenation and relaxing activities but had no supercoiling activity. The inhibitory effects of quinolones on the decatenation activity of topoisomerase IV were determined by quantitative electrophoresis with kinetoplast DNA as a substrate. The 50% inhibitory concentrations (IC50s) of levofloxacin, DR-3354, DU-6859a, DV-7751a, ciprofloxacin, sparfloxacin, and tosufloxacin against topoisomerase IV of S. aureus FDA 209-P were 2.3, 97, 0.45, 1.5, 2.5, 7.4, and 1.8 microg/ml, respectively, and were correlated well with their MICs. The IC50s of these drugs were from 2 to 20 times lower than those for the DNA gyrase. These results support genetic evidence that the primary target of new quinolones is topoisomerase IV in quinolone-susceptible strains of S. aureus. Three altered proteins of topoisomerase IV containing Ser-->Phe changes at codon 80 or Glu-->Lys changes at codon 84 of grlA, or both, were also purified. The inhibitory activities of quinolones against the topoisomerase IV which contained a single amino acid change were from 8 to 95 times weaker than those against the nonaltered enzyme. These results suggest that the mutations in the corresponding genes confer quinolone resistance.