A levofloxacin-sensitive strain of Escherichia coli (broth MIC: 0.0625 mg l(-1)) was grown in carbon-limited chemostat culture for 316 h (D=0.294 h(-1)). Hyperresistant strains isolated after 58 and 91 generations of culture retained a 16- to 47-fold increase in tolerance to levofloxacin during antibiotic-free serial batch and continuous culture (20 generations, glucose-limited, D=0.2 h(-1)). Isolates differed from the original strain in their maximum growth rates in the presence and absence of subinhibitory levels of levofloxacin, protein-banding profiles, and resistance to a range of antibiotics. Competition between resistant isolates and the original sensitive strain was studied in glucose-limited chemostat cultures (D=0.2 h(-1)). At levofloxacin concentrations less than 0.03 mg l(-1), the sensitive strain outcompeted resistant isolates and displaced them from the culture, whereas the reverse was true at higher concentrations. These results have clinical and environmental implications for those administering levofloxacin.