The mutant prevention concentration (MPC) for ciprofloxacin was determined for two Rhodococcus equi strains. The MPC for both strains was 32 mug/ml, which is above the peak serum concentration of ciprofloxacin obtainable by oral administration in humans. Nine single nucleotide changes corresponding to eight amino acid substitutions in the quinolone resistance-determining regions of DNA gyrase subunits A and B were characterized. Only mutants with amino acid changes in Ser-83 of GyrA were highly resistant (>or=64 microg/ml). Our results suggest that ciprofloxacin monotherapy against R. equi infection may result in the emergence of ciprofloxacin-resistant mutants.