In the treatment of Mycoplasma pneumoniae infection, erythromycin and clarithromycin, 14-membered macrolides, and azithromycin, a 15-membered macrolide, are usually considered to be the first-choice drugs. We found that ca. 20% of M. pneumoniae strains isolated from patients in Japan from 2000 to 2003 were macrolide resistant, with resistance resulting from an A-to-G transition at position 2063 or 2064 of the 23S rRNA gene. Given this, an alternative to macrolides is needed. Garenoxacin, a des-F(6)-quinolone exhibiting excellent activity against respiratory pathogens, is under development. In the present study, we examined the in vitro activity of garenoxacin against M. pneumoniae isolates, including macrolide-resistant strains, and compared it with those of other antibiotics. Using a broth microdilution method, we tested three macrolide-susceptible reference strains (FH, Mac, M129) and four macrolide-resistant clinical isolates (carrying A2063G or A2064G mutations in the 23S rRNA gene). The MICs of garenoxacin for all isolates were 0.016 to 0.031 μg/ml, two- to fourfold lower than those of gatifloxacin and 8- to 32-fold lower than those of levofloxacin and minocycline. These data indicate that garenoxacin may be a viable alternative to macrolides in the treatment of pneumonia caused by M. pneumoniae, including macrolide-resistant strains.