The phenotypic resistance of selected organisms to ciprofloxacin, levofloxacin, and trovafloxacin was defined as a MIC of > or =4 microg/ml. The dynamics of resistance were studied after single and sequential drug exposures: clinical isolates of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa were utilized. After a single 48-h exposure of a large inoculum to four times the initial MIC for the organism, the frequency of selection of resistant mutants of MSSA was greater for trovafloxacin than levofloxacin (P = 0.008); for E. cloacae, the frequency was highest for ciprofloxacin and lowest for levofloxacin and trovafloxacin; for S. marcescens, the frequency was highest for trovafloxacin and lowest for ciprofloxacin (P = 0.003). The results of serial passage experiments were analyzed both by the Kaplan-Meier product-limited method as well as by analysis of variance of mean inhibitory values. By both methods, MSSA and MRSA expressed mutants resistant to ciprofloxacin after fewer passages than were required for either levofloxacin or trovafloxacin. For the aerobic gram-negative bacilli, two general patterns emerged. Mutants resistant to trovafloxacin appeared sooner and reached higher mean MICs than did mutants resistant to levofloxacin or ciprofloxacin. Mutants resistant to ciprofloxacin appeared later and reached mean MICs lower than the MICs of the other two drugs studied. Even though individual strain variation occurred, the mean MICs were reproduced when the serial passage experiment was repeated using an identical panel of E. coli isolates. In summary, the dynamic selection of fluoroquinolone-resistant bacteria can be demonstrated in experiments that employ serial passage of bacteria in vitro.