Twenty-six clinical isolates of Mycobacterium abscessus resistant to amikacin were identified. Most isolates were from patients with posttympanostomy tube placement otitis media or patients with cystic fibrosis who had received aminoglycoside therapy. Isolates were highly resistant (MICs > 1024 microg/mL) to amikacin, kanamycin, gentamicin, tobramycin, and neomycin (all 2-deoxystreptamine aminoglycosides) but not to streptomycin. Sequencing of their 16S ribosomal (r) RNA revealed that 16 (94%) of 17 had an A-->G mutation at position 1408. In vitro-selected amikacin-resistant mutants of M. abscessus and Mycobacterium chelonae had the same resistance phenotype, and 15 mutants all had the same A-->G substitution at position 1408. Introducing an rRNA operon from Mycobacterium smegmatis with a mutated A-->G at this position into a single functional allelic rRNA mutant of M. smegmatis produced the same aminoglycoside resistance phenotype. These studies demonstrate this 16S rRNA mutation is responsible for amikacin resistance in M. abscessus, which has only one copy of the rRNA operon.