Fluoroquinolones are key antimicrobials in the treatment of more serious pneumococcal infections, especially for treating infections caused by penicillin-resistant strains. Increased use of newer fluoroquinolones should be accompanied by greater surveillance efforts to monitor resistance development as well as clonal dissemination. Streptococcus pneumoniae (n=551) collected from 3 medical centers in Italy (Catania, Genoa, and Rome) over a period of 4 years (2001-2004) as part of the SENTRY Antimicrobial Surveillance Program were susceptibility tested against >30 antimicrobials using reference broth microdilution methods. Mutations in the quinolone resistance-determining region (QRDR) were characterized by PCR and sequencing of parC, parE, and gyrA. Epidemiological relationships among levofloxacin-resistant isolates were determined using ribotyping, PFGE, serotyping combined with antimicrobial resistance profiles augmented by QRDR mutation patterns. Eighty-three (15.1%) isolates showed reduced susceptibility to ciprofloxacin (MIC>or=4 microg/mL) and 31 (5.6%) were resistant to levofloxacin. In 2001, all pneumococcal isolates were susceptible to levofloxacin and resistance rapidly emerged in all 3 medical centers in 2002. The overall rates of levofloxacin resistance in 2002-2004 were the following: Catania 10.9%, Genoa 3.3%, and Rome 6.5%. All resistant strains showed at least one mutation in parC and gyrA. Each isolate from Genoa with a unique resistance phenotype also showed distinct ribotype/PFGE, serotype, and QRDR mutation patterns. All isolates from Catania (n=19) showed an identical ribotype/PFGE pattern (333.3/A1); however, 3 distinct clusters could be identified based on further resistance phenotype, serotypes, and QRDR mutation pattern analysis. Two clusters were documented among isolates from Rome based on ribotype/PFGE. One isolate from Genoa shared ribotype/PFGE (333.3/A1) and serotype (9 not V) results with clusters from the other 2 institutions monitored, indicating clonal dissemination between the geographically diverse cities. In conclusion, fluoroquinolone resistance rates have increased among S. pneumoniae recovered in Italian medical centers evaluated by the SENTRY Program. Although resistance has emerged in many epidemiologically distinct strains, clonal dissemination seems to be a key contributing factor for increasing resistance to fluoroquinolones among pneumococci in this nation.