The aim of this study was to characterize quinolone resistance mechanisms in strains of Streptococcus pneumoniae with increased MICs of ofloxacin. These strains were also tested for their susceptibility to a battery of quinolone antimicrobial agents, including gemifloxacin. Of the S. pneumoniae isolates used, 27 were susceptible to ofloxacin, 18 intermediate and 48 resistant (ofloxacin MIC <4, 4 and >4 mg/L, respectively). In general, the ofloxacin-susceptible strains had no amino acid substitutions in GyrA, GyrB, ParC or ParE. Moderate increases in MIC were associated with substitutions in the quinolone resistance-determining region (QRDR) of ParC, while the highest MICs were found for strains that also had substitutions in the QRDR of GyrA. The most common substitutions were Ser79-->Phe in ParC and Ser81-->Phe in GyrA. Other substitutions were identified within the QRDR of ParC and outside the QRDR of ParC and ParE; these did not appear to affect susceptibility. The effects of antimicrobial efflux pumps were studied by determining MICs of a range of quinolones in the presence and absence of reserpine, an inhibitor of Gram-positive efflux pumps. Our results indicated that high-level resistance, caused entirely by efflux, was seen in a minority of ofloxacin-resistant S. pneumoniae strains. Testing the susceptibility of quinolone-resistant strains to gemifloxacin, ciprofloxacin, norfloxacin, ofloxacin and trovafloxacin revealed that gemifloxacin was least affected by this large variety of resistance mechanisms and was the only quinolone with MICs of < or =0.5 mg/L for all strains in this study. These results suggest that gemifloxacin is highly potent against S. pneumoniae and may also be effective against strains resistant to other quinolones.