In vitro antimicrobial activity of fleroxacin (6,8- difluoro-1-(2-fluoroethyl)-1, 4-dihydro-7- (4-methyl-1 -piperazinyl) -4-oxo-3-quinolinecarboxylic acid) and ofloxacin against representative pathogenic mycobacteria was evaluated by the agar dilution method, using 7H11 agar medium. Fleroxacin showed appreciable antimicrobial activity against Mycobacterium tuberculosis (MIC 90=6.25 mg/l), M. kansasii (MIC 90=3.13 mg/l), and M. fortuitum (MIC 90=6.25 mg/l), whereas M. marinum, M. scrofulaceum, M. avium, M. intracellulare, and M. chelonae were highly resistant to the agent. The activity of fleroxacin was comparable to that of ofloxacin. Fleroxacin showed antimicrobial activity against M. intracellulare phagocytosed in murine peritoneal macrophages at a concentration of 10 mg/l in the culture medium, but its activity was considerably lower than that of ofloxacin. On the other hand, the therapeutic activity of fleroxacin against M. fortuitum infection induced in mice was higher than that of ofloxacin. Neither fleroxacin nor ofloxacin was efficacious against M. intracellulare infection. Fleroxacin significantly depressed the growth of M. leprae in the mouse footpad.