We identified fluoroquinolone-resistant Streptococcus pneumoniae strains among 670 clinical isolates isolated from 1999 to 2003 in Hokkaido prefecture, Japan. All eleven stains were resistant to ciprofloxacin and levofloxacin. Furthermore, ten strains were also resistant to fluoroquinolones that are more effective with gram-positive bacteria, namely tosufloxacin, sparfloxacin, and gatifloxacin. Nucleotide sequence analysis of the quinolone-resistance determining region (QRDR) of the quinolone target genes coding for topoisomerase IV subunits (parC and parE) and DNA gyrasesubunits (gyrA and gyrB). Eight stains, which showed higher resistance, had resistance mutations in two genes (gyrA and parC, or gyrA and parE), and other three strains had one resistance mutation in parC. The mutation patterns were varied between the strains. Data from random amplified polymorphic DNApolymerase chain reaction (RAPD-PCR) indicated that eleven strains were identified as ten independent clones. Lines of evidence indicated that genetic mutations leading to fluoroquinolone resistance occur sporadically rather through the spreading of a particular resistant strain. Notably, the fluoroquinolone-resistant strains were only isolated from adults, particularly from patients more than 60 years of age (9/60 strains; 15.0%). Resistant strains were not found in 574 strains isolates from patients under 20 years of age. This may be due to the fact that fluoroquionolones other than norfloxacin are not applicable to children in Japan.