Eighteen quinolone-resistant isolates of Escherichia coli were selected by exposing ten clinical isolates to increasing concentrations of norfloxacin and lomefloxacin. The mutant isolates showed a multiple-antibiotic-resistance phenotype. All of them contained single mutations in gyrA consisting of the substitution of Ser-83→Leu (n = 14), Val (n = 1) or Ala (n = 1) and the substitution of Asp-87→Asn (n = 2). Only one concomitant mutation in parC (Ser-80→Arg) was detected. Four parent isolates exhibited a single mutation in gyrA which required ≤ 1 mg/L of norfloxacin to be inhibited. Fluoroquinolone resistance, in the 18 quinolone-resistant mutants, was a result of mutations affecting DNA gyrase plus decreased fluoroquinolone uptake. This latter mechanism of resistance was a combined effect of an absence of OmpF and an increase in active efflux in eight isolates, or an increased active efflux alone in the remaining ten selected mutants.