An 81-year-old woman had pneumonia caused by Streptococcus pneumoniae (levofloxacin Etest minimum inhibitory concentration [MIC] 1.5 μg/ml) and was treated with intravenous gatifloxacin 200 mg/day. After 3 days of therapy, repeat sputum cultures were positive for S. pneumoniae, which was resistant to levofloxacin (Etest MIC > 32 μg/ml). The isolate obtained before therapy showed a preexisting parC mutation of aspartic acid-83 to asparagine (Asp83→Asn), and the isolate obtained during therapy showed an acquired gyrA mutation from serine-81 to phenylalanine (Ser81→Phe) and a second parC mutation from lysine-137 to Asn (Lys137→Asn). Both isolates were the same strain, as determined with pulsed-field gel electrophoresis. This case demonstrates the potential for resistance to emerge during 8-methoxy fluoroquinolone therapy for fluoroquinolone-susceptible S. pneumoniae with a preexisting parC mutation. Additional clinical failures with a fluoroquinolone may occur unless these first-step parC mutants can be identified to assist clinicians in selecting appropriate antimicrobial therapy.