The purpose of this study was to establish a standardized protocol for second-line antimicrobial suscepti-bility testing of Mycobacterium tuberculosis using the Bactec MGIT 960 system in Canadian laboratories. Four Canadian public health laboratories compared the susceptibility testing results of 9 second-line antimicrobials between the Bactec 460 and Bactec MGIT 960 systems. Based on the data generated, we have established that the Bactec MGIT 960 system provides results comparable to those obtained with the previous Bactec 460 method. The critical concentrations established for the testing of the antimicrobials used are as follows: amikacin, 1 g/ml; capreomycin, 2.5 g/ml; ethionamide, 5 g/ml; kanamycin, 2.5 g/ml; linezolid, 1 g/ml; moxifloxacin, 0.25 g/ml; ofloxacin, 2 g/ml; p-aminosalicylic acid, 4 g/ml; rifabutin, 0.5 g/ml. The Public Health Agency of Canada (PHAC) publishes yearly statistics on the antimicrobial resistance patterns of all laboratory-isolated Mycobacterium tuberculosis strains in Can-ada. From 2000 to 2010, the number of M. tuberculosis isolates resistant to one or more of the first-line antimicrobials has varied between 8.0 % and 11.0 % of all tuberculosis (TB) cases per year in Canada (15). Between 0.9 % and 1.6 % of these