We describe 66 ciprofloxacin-nonsusceptible Streptococcus pyogenes isolates recovered from colonized and infected children. The ParC S79A substitution was frequent and associated with the emm6/sequence type 382 (emm6/ST382) lineage. The ParC D83G substitution was detected in two isolates (emm5/ST99 and emm28/ST52 lineages). One isolate (emm89/ST101) had no quinolone resistance-determining region codon substitutions or other resistance mechanisms. Five of 66 isolates were levofloxacin resistant. Although fluoroquinolones are not used in children, they may be putative disseminators of fluoroquinolone-nonsusceptible strains in the community.Streptococcus pyogenes clinical isolates with reduced susceptibility to fluoroquinolones (1,3,8,14,16,18,27,29) or with high-level resistance (15,16,(23)(24)(25)28) have been described previously, and the reduced susceptibility to fluoroquinolones is mediated by point mutations in the quinolone resistancedetermining region (QRDR) of the parC gene (3, 16, 18) whereas high-level resistance has been associated with mutations in the QRDRs of both parC and gyrA genes (23, 28). To the best of our knowledge, there are no reports documenting the prevalence and characterization of fluoroquinolone-nonsusceptible S. pyogenes associated with asymptomatic colonization. Since 1999 and 2000, we have been collecting S. pyogenes isolates from pediatric patients from different clinical origins and from carriers for the surveillance of antimicrobial susceptibility and for the epidemiological characterization of the isolates. This study aimed to describe the prevalence of ciprofloxacin-nonsusceptible S. pyogenes isolates from colonized and infected Portuguese children from 1999 to 2006 and to characterize the associated clones and resistance mechanisms.Strains and antibiotic susceptibility. A total of 1,354 nonduplicated S. pyogenes isolates were collected from children in the Lisbon area in Portugal; 901 were associated with asymp-