Linezolid is an oxazolidinone available as an oral drug which has activity against most gram-positive bacteria. However, few species of the genus Mycobacterium have been studied. We tested 249 clinical isolates and 10 reference strains of rapidly growing mycobacteria for susceptibility to linezolid by broth microdilution. Clinical species included the Mycobacterium fortuitum group (n = 74), M. abscessus (n = 98), M. chelonae (n = 50), M. mucogenicum (n = 10), and M. fortuitum third biovariant complex (10). The modal MIC for M. mucogenicum was 1.0 microg/ml, and the MIC at which 90% of the isolates tested are inhibited (MIC(90)) was 4 microg/ml; the modal MIC for the M. fortuitum group was 4 microg/ml, and the MIC(90) was 16 microg/ml; the modal MIC for the M. fortuitum third biovariant complex was 4 microg/ml, and the MIC(90) was 8 microg/ml; the modal MIC for M. chelonae was 8 microg/ml, and the MIC(90) was 16 microg/ml; and the modal MIC for M. abscessus was 32 microg/ml, and the MIC(90) was 64 microg/ml. Based on peak levels of linezolid in serum of 15 to 20 microg/ml, we propose the following broth MIC breakpoints for these species: susceptible, < or = 8 microg/ml; moderately susceptible, 16 microg/ml; and resistant, > or =32 microg/ml). These studies demonstrate the excellent potential of linezolid for therapy of rapidly growing mycobacteria.