The Amplicor Mycobacteria, a PCR-based assay, is a rapid test for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium intracellulare in clinical samples. To estimate the reliability and reproducibility of the method, a cooperative blind study was conducted among 9 laboratories. Materials used for testing consisted of 105 sputum and 30 water samples containing known numbers of M. bovis BCG, M. avium, M. intracellulare, and samples without bacteria. Only 2 out of the 9 laboratories correctly identified the presence or absence of mycobacterial DNA in all 135 samples. In sputum samples, 6 out of the 9 laboratories detected mycobacterial DNA in all positive samples, and 4 out of the 9 laboratories correctly reported the absence of DNA in the negative samples, indicating the need for good laboratory practice and development of reference reagents to monitor the performance of the whole study, including pretreatment of clinical samples. The main problem was lack of specificity rather than lack of sensitivity. From about half of the laboratories, false-positive results were reported, however, the ratio was below 6%; 1% (1/106 sputum samples) in 3 laboratories, 1.9% (2/105) in 2 laboratories, and 5.7% (6/105) in one laboratory, respectively. These results indicate that the Amplicor Mycobacteria is quite useful for a rapid diagnosis of tuberculosis.