We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (Х99%) and specificity (100%) and an appropriate detection limit (10 5 CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories.Tuberculosis is a global problem and the single most common cause of death from any bacterial agent (15,22). Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) are different clinically, so prompt detection, isolation, and discrimination are essential for appropriate management (3, 7).The MPT64 protein is highly specific for M. tuberculosis complex, including M. tuberculosis, Mycobacterium africanum, Mycobacterium bovis, and some, although not all, substrains of M. bovis BCG (1,6,16,18,23), and can be detected in culture isolates and biopsy samples (1,7,17,18,20). Recently, Standard Diagnostics (SD, Yongin, Korea) developed a simple and rapid assay using a mouse monoclonal anti-MPT64 antibody to discriminate between M. tuberculosis complex and NTM by immunochromatography. Mouse monoclonal anti-MPT64 antibodies (SD Bioline TB Ag MPT64; SD) are immobilized on a nitrocellulose membrane as the capture material. Another antibody, which recognizes another epitope of MPT64 and has been conjugated with colloidal gold particles, is used for antigen capture and detection in a sandwich-type assay.We evaluated the clinical usefulness of the kit using mycobacteria and other organisms. To determine specificity, 137 bacterial isolates (68 species), 20 fungal isolates (10 species), 53 reference mycobacterial isolates (40 species), and 51 NTM isolates from clinical samples were tested (Tables 1 to 3). To determine sensitivity, 159 M. tuberculosis complex strains grown on 3% Ogawa medium (isolated at Pusan National University Hospital), 60 strains from Bactec MGIT 960 culture tubes (isolated at Kosin University Gospel Hospital), and one reference strain, M. tuberculosis H37Rv, were tested. All bacterial, fungal, and mycobacterial isolates were stock cultures that had been kept in a Ϫ4°C refrigerator or a Ϫ72°C deep freezer for as long as 18 months. The cultured mycobacteria were identified by acid-fast bacillus stain, nucleic acid amplification, and DNA microarray (10,14). Finally, to determine the detection limit, a series of diluted suspensions of M. tuberculosis H37Rv were inoculated onto Middlebrook 7H10 agar and the resulting colonies were counted (19).