The potential for resistance development in Streptococcus pneumoniae secondary to exposure to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin at various levels was examined at high inoculum (10(8.5) to 10(9) log10 CFU/ml) over 96 h in an in vitro pharmacodynamic (PD) model using two fluoroquinolone-susceptible isolates. The pharmacokinetics of each drug was simulated to provide a range of free areas under the concentration-time curves (fAUC) that correlated with various fluoroquinolone doses. Potential first (parC and parE)- and second-step (gyrA and gyrB) mutations in isolates with raised MICs were identified by sequence analysis. PD models simulating fAUC/MICs of 51 and<or=60, 34 and 37, <or=82 and<or=86, and<or=24 for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, respectively, against each isolate were associated with first-step parC (S52G, S79Y, and N91D) and second-step gyrA (S81Y and S114G) mutations. For each fluoroquinolone a delay of first- and second-step mutations was observed with increasingly higher fAUC/MIC ratios and recovery of topoisomerase mutations in S. pneumoniae was related to the fAUC/MIC exposure. Clinical doses of gatifloxacin, gemifloxacin, and moxifloxacin exceeded the fAUC/MIC resistance breakpoint against wild-type S. pneumoniae, whereas those of levofloxacin (500 and 750 mg) were associated with first- and second-step mutations. The exposure breakpoints for levofloxacin were significantly different (P<0.001) from those of the newer fluoroquinolones gatifloxacin, gemifloxacin, and moxifloxacin. Additionally, moxifloxacin breakpoints were significantly lower (P<0.002) than those of gatifloxacin. The order of resistance development determined from fAUC/MIC breakpoints was levofloxacin>gatifloxacin>moxifloxacin=gemifloxacin, which may be related to structural differences within the class.