The ability to determine various mycobacterial species rapidly and precisely is important for the control and prevention of mycobacterial infections in the human population. In the present study, we evaluated the Roche Amplicor Mycobacterium tuberculosis test and the Amplicor M. avium/M. intracellulare test, as well as a multiplex PCR, for diagnosis of mycobacterial infections using 86 clinical (sputum and bronchial washing) and culture specimens from 50 human patients. The Roche Amplicor M. tuberculosis test and the Amplicor M. avium/M. intracellulare test appeared to be highly sensitive and specific for direct detection of M. tuberculosis and M. avium/M. intracellulare, respectively, from clinical specimens. The use of culture derivatives was not necessary. Nonetheless, the Roche Amplicor tests had limited value in identifying other mycobacterial species as these mycobacteria invariably displayed negative reactions in the assays. The multiplex PCR, on the other hand, was capable of differentiating M. tuberculosis, M. avium and M. intracellulare from other mycobacterial species using culture specimens at relatively low cost. However, the multiplex PCR generated inconsistent results when clinical samples were used. Only 42 (48.8%) of the 86 sputum and bronchial washing specimens showed clear, specific electrophoretic bands in the multiplex PCR. Future optimisation of a hybridisation-based detection system is essential for enhanced identification of mycobacteria directly from clinical specimens by the multiplex PCR.