The MICs of 13 antimicrobial agents including seven fluoroquinolones (ciprofloxacin, levofloxacin, sparfloxacin, grepafloxacin, gatifloxacin, moxifloxacin and clinafloxacin) for Streptococcus pneumoniae isolates obtained from all regions of Hong Kong in the year 2000 were determined by the Etest. Overall, 39.4% of 180 isolates were susceptible to penicillin, 11.7% were intermediate and 48.9% were resistant. The overall prevalence of fluoroquinolone non-susceptibility (levofloxacin MIC 4 mg/L) was 13.3% but increased to 27.3% among the penicillin-resistant isolates. For the fluoroquinolone non-susceptible isolates, within-class cross-resistance was common. For the fluoroquinolone non-susceptible isolates, the median MICs of clinafloxacin, gatifloxacin, moxifloxacin, sparfloxacin and grepafloxacin were, respectively, six-, 24-, 32- 84- and 128-fold higher than those for the susceptible isolates. All fluoroquinolone non-susceptible strains were derived from adults. The prevalence of fluoroquinolone resistance was higher in isolates from older patients (17.1% among those 65 years of age versus 9.1% among those 18–64 years of age, P < 0.001) and from adults with chronic obstructive pulmonary disease (24.6% versus 9.3%, P = 0.01). All fluoroquinolone non-susceptible strains were non-susceptible to penicillin (MIC range 2–4 mg/L), cefotaxime (MIC range 1–4 mg/L) and erythromycin (MIC range 4–256 mg/L). The fluoroquinolone non-susceptible isolates were genetically related to the Spain23F-1 clone when analysed by pulse-field gel electrophoresis and multilocus sequence typing. In conclusion, a rapid increase in the prevalence of fluoroquinolone resistance among S. pneumoniae was found in Hong Kong. Typing analysis suggests that this is due to the pan-regional dissemination of a fluoroquinolone-resistant variant (designated Hong Kong23F-1) of the globally distributed Spain23F-1 clone.