By use of a PCR-restriction fragment length polymorphism assay, we screened 496 levofloxacin-susceptible invasive pneumococcal strains (MIC<or=2 mg/liter) for quinolone resistance-determining region mutations known to confer fluoroquinolone resistance. Among those with a levofloxacin MIC of 2 mg/liter, 16.2% of isolates recovered from nursing home residents and 6.4% from non-nursing home residents had first-step mutations.