Sparfloxacin (SPFX), a new quinolone, was studied its in vitro and in vivo activities against various mycobacteria, especially Mycobacterium intracellulare. SPFX exhibited a potent in vitro activity against M. tuberculosis, M.kansasii and M.fortuitum with MIC90 values of 0.2, 6.25 and 1.6 micrograms/ml, respectively, and the potency of SPFX was higher than that of ofloxacin (OFLX). M.marinum, M.scrofulaceum, M.avium, M.intracellulare, M.chelonae (subsp. abscessus and chelonae) were resistant to SPFX. SPFX inhibited the growth of M. intracellulare in 7H9 broth when added at the concentration of 0.2 microgram/ml and rapidly killed the organisms at the dose of 1 microgram/ml. The activity of SPFX was higher than that of OFLX. SPFX exhibited a greater antimicrobial activity against M.intracellulare phagocytosed by murine peritoneal macrophages than did OFLX. SPFX exhibited a weak therapeutic activity against M.intracellulare infection induced in mice, on the basis of the rate of bacterial elimination in the host lungs and spleen, but such an efficacy was not noted for OFLX. SPFX combined with rifampicin (RFP), or in combination with RFP and kanamycin yielded a slightly increased therapeutic efficacy, based on the degree of pulmonary gross lesions in host animals or CFU of organisms in the lungs and spleen.