BACKGROUND: Microscopic examination of sputum smears for acid-fast bacilli (AFB) is the most important and rapid diagnostic test for pulmonary tuberculosis. However, the AFB observed on the smear may represent either M. tuberculosis or nontuberculous mycobacteria (NTM). This study examined the clinical usefulness of a polymerase chain reaction test for M. tuberculosis (TB-PCR) for the differentiation of pulmonary tuberculosis and NTM lung disease in patients with smear-positive sputums in a tertiary hospital in Korea. MATERIAL AND METHODS: From January, 2003 to December, 2003, 826 AFB smear-positive and culture-positive sputum specimens were collected from 299 patients. RESULTS: NTM were recovered from 26.6% (220/826) of the smear-positive sputum specimens and 23.4% (70/299) of the patients with smear-positive sputum. All the patients with isolated NTM had clinically significant NTM lung disease; 38 patients (54.3%) had M. avium and 26 patients (37.1%). had M. abscessus. In the patients with pulmonary tuberculosis, 78.7% of the patients (74/94) showed TB-PCR positivity, and all the patients with NTM lung disease showed negative results on the TB-PCR test (p<0.001). A positive result of the TB-PCR test on the sputum or bronchial washing fluid specimens was able to predict pulmonary tuberculosis with 88.4% sensitivity, 100% specificity, a 100% positive predictive value and a 79.7% negative predictive value for the patients with smear-positive sputum. CONCLUSION: The TB-PCR test for sputum specimens or bronchial washing fluid specimens could be useful for differentiating pulmonary tuberculosis and NTM lung disease for the patients with smear-positive sputum in Korea.