Background :A total of 50 strains of mycobacteria, including 11 reference strains(9 atypical mycobacteria and 2 M. tuberculosis strains) and 39 clinical isolated strains was investigated for a single tube heat stable acid phosphatase, 68`C catalase and niacin tests. The reference strains of 9 atypical mycobacteria were positive in a single tube heat stable acid phosphatase test and/or 68% catalase activity, but 2 M. tuberculosis strains were negative in both activities. Nine strains of clinical isolates were positive in heat stable acid phosphatase and/or 68`C catalase tests, and negative in niacin test, which were confirmed as atypical mycobacteria, while 30 strains of clinical isolates were negative in heat stable acid phosphatase and 68% catalase tests, and positive in niacin test (6 strains were not distinct), which were confirmed as M. tuberculosis. We conculude that the single tube heat stable acid phosphatase and 68oC catalase tests are very simple, rapid and accurate methods for the differentiation of atypical mycobacteria from M. tuberculosis. Method : Result : Conclusion :