In 2000, the Canadian mycobacteriology proficiency testing program was updated and expanded to improve proficiency and standardize the main activities and testing performed in Canadian clinical mycobacteriology laboratories. The program was divided into 2 phases and included samples for acid-fast microscopy, species identification, susceptibility testing for Mycobacterium tuberculosis and nontuberculous mycobacteria, nucleic acid amplification for the detection of M. tuberculosis complex in clinical samples, and restriction fragment length polymorphism (RFLP) typing of M. tuberculosis. Results of participating laboratories are given for a 3-year period (2000-2002). Canadian laboratories exhibited a high level of proficiency in both phases of the program. Improvements were demonstrated in susceptibility testing of M. tuberculosis, and standardization was achieved with Mycobacterium avium complex susceptibility testing. Laboratories demonstrated proficiency in determining unique and identical strains of M. tuberculosis using RFLP; however, difficulties occurred with interlaboratory reproducibility.