Background: Nontuberculous mycobacteria (NTM) are frequently isolated from environmental sources and can cause contamination of respiratory specimens. This study was performed to evaluate the clinical significance of a single isolation of pathogenic NTM from sputum specimens. Methods: We identified single NTM isolates from sputum specimens at the Samsung Medical Center (Seoul, South Korea) obtained from January 2002 to December 2003. If the patients had compatible clinical and radiographic findings and the same pathogenic NTM species were isolated from subsequent sputum examinations, the patients were diagnosed with NTM lung disease. Results: We identified 190 patients with a single sputum culture of pathogenic NTM: 120 patients had MAC (67 with M. avium and 53 with M. intracellulare), 62 had M. abscessus, and 8 had M. kansasii. During the follow-up period, 26 patients (14%) had an additional positive culture of the same NTM species and met the ATS diagnostic criteria. Sixteen patients (13%) with a single isolate of MAC, 10 patients (16%) with a single isolate of M. abscessus and none patients with a single isolate of M. kansasii were diagnosed with NTM lung disease. The median duration from the first sputum culture to the second was 30 months (IQR, 15-46 months). The common CT findings were bronchiectasis (26/26; 100%) and bronchiolitis (20/26; 77%). Conclusion: Patients with a single sputum culture positive for pathogenic NTM, particularly those with underlying bronchiectasis, might benefit from long-term follow-up to confirm or exclude NTM lung disease.