The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and The rapid identification of Mycobacterium spp. from smearpositive sputum samples is very important from clinical viewpoints. This is because, in addition to increased clinical infections due to Mycobacterium tuberculosis complex (MTBC) organisms, the percentage of infections with nontuberculous mycobacteria (NTM) also has been increasing recently (1, 6-8, 11, 16, 20, 21, 31), especially among immunocompromised patients (11). Strategies used for the clinical management of patients with MTBC or NTM infections are different. Patients who are suspected to have MTBC infections have to receive proper medication or even be placed in an isolation room immediately. Therefore, the correct and rapid identification of MTBC and NTM organisms represents a clinical emergency that should not be underestimated.The traditional diagnosis of mycobacterial infections from sputum samples in the mycobacterial laboratory is based primarily on demonstrating the presence of the acid-fast bacilli (AFB) in the smear, followed by a positive culture and the testing of the physiological/biochemical identification of the isolate (19). This approach has a number of defects, including that it is time-consuming, has low sensitivity, and has poor discrimination between closely related NTM species (29). High-performance liquid chromatography is an alternative approach for the identification of mycobacteria, and this approach can identify more than 50 different species (9); however, a concentration of more than 10 6 bacteria per ml is required. Recently, a paranitrobenzoic acid assay has been applied directly to clinical samples as a rapid screening assay for the detection of and differentiation between MTBC and NTM (32). Nevertheless, this approach does not allow further NTM species differentiation and has a long incubation time (3 weeks) before the results can be read.Recently, the development of PCR-based methods for the rapid identification and differentiation of mycobacterial organisms has significantly improved the diagnosis efficiency in terms of both sensitivity and specificity (3,5,12,14,17,22,24,26,27,30). We previously developed a multiplex nested PCR combined with lateral-flow technology for the rapid diagnosis of M. tuberculosis and MTBC isolates and the differentiation of these organisms from NTM organisms (28). In addition, a multiplex PCR system for the rapid detection and differentiation of MTBC mem...