The in vitro activities of isepamicin against 117 Mycobacteria abscessus, 48 Mycobacterium fortuitum, and 20 Mycobacterium chelonae isolates were evaluated by a microdilution test. Isepamicin MIC(90)s were < or =16 microg/ml for the three species. Isepamicin was as active as amikacin and kanamycin and more active than tobramycin, capreomycin, gentamicin, and streptomycin.