The mutations in the quinolone resistance-determining region of the gyrA and gyrB genes from 27 clinical isolates of Escherichia coli with a range of MICs of ciprofloxacin from 0.007 to 128 μg/ml and of nalidixic acid from 2 to >2,000 μg/ml were determined by DNA sequencing. All 15 isolates with ciprofloxacin MICs of ≥1 μg/ml showed a change in Ser-83 to Leu of GyrA protein, whereas in clinical isolates with a MIC of ≥8 μg/ml (11 strains), a double change in Ser-83 and Asp-87 was found. All isolates with a MIC of nalidixic acid of ≥128 μg/ml showed a mutation at amino acid codon Ser-83. Only 1 of the 27 clinical isolates of E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. A change in Ser-83 is sufficient to generate a high level of resistance to nalidixic acid, whereas a second mutation at Asp-87 in the A subunit of DNA gyrase may play a complementary role in developing the strain's high levels of ciprofloxacin resistance.