Twenty-three clinical strains of Chlamydia trachomatis were isolated from men with chlamydial nongonococcal urethritis and examined for the presence of fluoroquinolone resistance-associated alterations in GyrA and ParC. The minimum inhibitory concentrations (MICs) of fluoroquinolones, erythromycin, and tetracycline were determined for 6 of the 23 isolates. In 12 of the 23 isolates, a single amino-acid change was found in GyrA, and in 1 isolate, two amino acids were changed. In all 23 isolates, an Arg-83-to-Gly substitution was observed in ParC, and in 3 isolates, an additional amino-acid change was found. Some changes occurred within the quinolone resistance-determining regions (QRDRs) of GyrA and ParC, but not at positions critical for fluoroquinolone resistance. Of the 6 isolates for which MICs of the agents were determined, 1 isolate had a Cys-66 --> Arg substitution in GyrA, and all had the Arg-83 --> Gly substitution in ParC. However, all 6 isolates were susceptible to fluoroquinolones. First-pass urine specimens were obtained from two men who were positive for C. trachomatis after levofloxacin treatment, and the gyrA and parC genes of C. trachomatis were amplified by polymerase chain reaction (PCR) and examined for fluoroquinolone resistance-associated mutations. Pre- and post-treatment C. trachomatis persisting in each of them had identical amino-acid sequences in the QRDR of GyrA and ParC. Further, the substitutions found in GyrA and ParC were not located at positions critical for fluoroquinolone resistance. The present study suggests that fluoroquinolone resistance-associated alterations in GyrA and ParC may be uncommon in clinical strains of C. trachomatis.