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Background: The isolation rate of nontuberculous mycobacteria (NTM) in clinical laboratories and the incidence of NTM infections are on the increase recently in Korea, but there have been only a few studies that reveal the general aspect of NTM isolation or species distribution. Therefore, this study was performed to examine the isolation rate of NTM, species identification, and the clinical significance of mycobacterial cultures., Methods: From August 2004 to May 2005, we examined mycobacterial isolates by AccuProbe test to differentiate NTM from Mycobacterium tuberculosis complex. NTM was then identified by polymerase chain reaction-restriction fragment length analysis (PCR-RFLP)., Results: A total of 6,742 specimens from 2,784 patients were requested for mycobacterial culture. Mycobacteria were isolated from 776 specimens (11.5%). The isolation rates of NTMs among the total culture positive specimens and culture positive sputum specimens were 24.4% (189/776) and 25.3% (169/667), respectively. Fourteen species of NTM identified in 172 of the 175 specimens tested included M. avium (39.0%), M. intracellulare (22.7%), and M. abscessus (19.8%)., Conclusions: Using AccuProbe tests and PCR-RFLP method for mycobacterial cultures processed in a clinical laboratory, we were able to identify NTMs to the species level. The isolation rate of NTM in this study was similar to that reported in past studies in Korea. In addition, we found that some of the NTMs isolated in this study could cause pulmonary diseases.