Aims: To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium-intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. Methods and Results: The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty-two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan-mycobacterial and 133 bp species-specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan-mycobacterial bands of approx. 300-350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96.4% of smear-positive specimens and 70.2% of smear-negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2.0%). Conclusion, Significance and Impact of the Study: Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.