The polymerase chain reaction (PCR) was used to identify mycobacterial DNA sequences in uncultured clinical specimens. Two oligonucleotide primers derived from the sequence of a gene that codes for the 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA from all 11 species of mycobacteria tested. Amplified DNAs of nontuberculosis mycobacteria were found to be approximately 20 to 40 bases shorter than those from M. tuberculosis and Mycobacterium bovis BCG. DNA equivalent to that present in as few as 40 M. tuberculosis cells either alone or in the presence of DNA equivalent to that in 10(6) human cells could be detected. Results from analysis of cultured bacteria and clinical specimens showed PCR was sensitive and specific both in detecting mycobacteria and in differentiating M. tuberculosis and BCG from other species of mycobacteria. The PCR method with the primers reported here may become a useful tool in the early and rapid detection of mycobacterial infections in uncultured clinical specimens.