Garenoxacin showed the most potent chlamydial activity against Chlamydia trachomatis D/UW-3/Cx among three tested quinolones and azithromycin. The DNA gyrase genes, gyrA and gyrB, of C. trachomatis D/UW-3/Cx were cloned and the GyrA and GyrB subunits of DNA gyrase protein were separately expressed as histidine-tagged proteins in Escherichia coli. The mean 50% inhibitory concentration (IC(50)) of garenoxacin against the supercoiling activity of C. trachomatis D/UW-3/Cx gyrase was 2.9 ± 0.4 μg/ml, which was the most potent inhibitory activity against DNA gyrase among the quinolones tested in this study. At an extracellular concentration of 0.5 μg/ml, the cellular-to-extracellular concentration ratio of garenoxacin was 15.3 ± 1.3, equivalent to that of moxifloxacin and greater than that of levofloxacin. In a time-kill experiment, after exposure to garenoxacin at a concentration of 0.5 μg/ml at 0-6, 5-11, and 24-30 h after infection, the percentages of recoverable chlamydial inclusion-forming units were 11.1 ± 3.3, 0.6 ± 0.1, and 2.6 ± 0.5%, respectively. On transmission electron microscopy observation, after exposure to garenoxacin at 24-30 h after infection, some C. trachomatis elementary bodies remained in the inclusion body; however, the reticulate bodies were completely disrupted. In conclusion, garenoxacin is expected to be a useful quinolone in the treatment of infectious diseases caused by C. trachomatis.