g Ureaplasma spp. cause several disorders, such as nongonococcal urethritis, miscarriage, and preterm delivery with lung infections in neonates, characterized by pathological chorioamnionitis in the placenta. Although reports on antibiotic resistance in Ureaplasma are on the rise, reports on quinolone-resistant Ureaplasma infections in Japan are limited. The purpose of this study was to determine susceptibilities to five quinolones of Ureaplasma urealyticum and Ureaplasma parvum isolated from perinatal samples in Japan and to characterize the quinolone resistance-determining regions in the gyrA, gyrB, parC, and parE genes. Out of 28 clinical Ureaplasma strains, we isolated 9 with high MICs of quinolones and found a single parC gene mutation, resulting in the change S83L. Among 158 samples, the ParC S83L mutation was found in 37 samples (23.4%), including 1 sample harboring a ParC S83L-GyrB P462S double mutant. Novel mutations of ureaplasmal ParC (S83W and S84P) were independently found in one of the samples. Homology modeling of the ParC S83W mutant suggested steric hindrance of the quinolone-binding pocket (QBP), and de novo prediction of peptide structures revealed that the ParC S84P may break/kink the formation of the ␣4 helix in the QBP. Further investigations are required to unravel the extent and mechanism of antibiotic resistance of Ureaplasma spp. in Japan.Ureaplasma spp. are among the smallest self-replicating organisms, in terms of both genome size and cellular dimensions. They lack cell walls and are thus resistant to penicillin and other -lactams. These organisms exist in association with eukaryotic cells, mainly colonizing mucosal surfaces of the respiratory and urogenital tracts (1, 2). Ureaplasma spp. are common inhabitants of the lower genital tract and can be isolated from 40% to 80% of women of child-bearing age (1, 3). Ureaplasma spp. have been associated with a range of pathologies, including nongonococcal urethritis (NGU), miscarriage, preterm delivery, neonatal pneumonia, and chronic lung disease in preterm neonates (4, 5). Many reports have suggested that Ureaplasma parvum and/or Ureaplasma urealyticum may be associated with urogenital infections, infertility, and adverse pregnancy outcomes (6). Regarding the management of ureaplasmal infections, only a limited number of reports are available on the surveillance of antimicrobial resistance in clinical Ureaplasma strains, which is crucial for providing therapy empirically (6). The treatment of ureaplasmal infections is limited to tetracyclines, macrolides, and quinolones (5, 7). Resistance to all the three antibiotic classes in clinical Ureaplasma isolates has been documented, with unique nucleic acid substitutions identified as potential molecular mechanisms for each (5,(8)(9)(10).Quinolones are used for treating urogenital infections and interact in bacteria with the type II topoisomerases DNA gyrase and topoisomerase IV, both of which are composed of two A and two B subunits; these subunits are encoded by the gyrA and gyrB genes for DNA...