Mycobacterial antigen MPB64 has been identified as a Mycobacterium tuberculoisis complex-specific secretory protein since 1984. Recently, a simple culture confirmation test for M. tuberculosis complex has been developed by using lateral flow immunochromatographic assay (ICA) to detect MPB64 with anti-MPB64 monoclonal antibody. The current multicenter study evaluated the performance of an ICA slide test for MPB64 antigen in the clinical setting. Primary positive cultures from clinical specimens, as well as stock cultures, were tested. Approximately 100 μl of positive liquid culture medium or suspension made from colonies on solid medium was placed into the test well of the plastic slide devise, and the test was read after 15 min. No processing or instrumentation was required. A total of 304 mycobacterial isolates consisting of M. tuberculosis complex (171 isolates) and mycobacteria other than M. tuberculosis (MOTT) complex (133 isolates) belonging to 18 different species were tested. Growth in liquid media (Mycobacteria Growth Indicator Tube [MGIT] and Radiometric 12B), as well as in solid (Löwenstein-Jensen and Middlebrook 7H10 agar) media, was evaluated. Results were compared with those obtained with nucleic acid-based and/or high-pressure liquid chromatography identification. All MOTT were found to be negative on the ICA slide with no cross-reaction. All M. tuberculosis and M. africanum cultures were found to be positive, whereas the results of M. bovis and M. bovis BCG cultures were variable since some of the BCG strains are known to lack MPB64 antigen production. The results did not change with prolonged storage of cultures. This low-tech rapid test with high sensitivity and specificity could provide an alternative to currently available identification methods, particularly for recently introduced nonradiometric liquid culture systems such as MGIT.