Aminoglycoside acetyltransferase was detected in Mycobacterium kansasii and M. fortuitum but not in M. avium-M. intracellulare when they were screened by a radioassay. Aminoglycoside phosphotransferase and nucleotidyltransferase activities were absent from all three species tested. Acetyltransferases from both M. kansasii and M. fortuitum displayed relatively high K(m)s, all at the millimolar level, for substrates including tobramycin, neomycin, and kanamycin A. The K(m) of each substrate was well above the corresponding maximum achievable level in serum. The low affinities of these enzymes for their substrates suggested that drug modification in vivo was very unlikely. Among the various substrates tested, no apparent positive correlation was found between substrate affinity and resistance level. The presence of aminoglycoside-modifying enzymes in these mycobacterial species was therefore not shown to confer resistance to aminoglycosides.