Known DNA sequences coding for the 16S rRNAs of 14 slowly growing Mycobacterium species were analyzed. Three sets of primers were synthesized: MAV and MIN, for M. avium and M. intracellulare, respectively, and MYCOB, for the slowly growing mycobacteria. Whole-cell DNAs of 14 reference species were extracted and amplified by PCR with the MYCOB, MAV, and MIN primers. The MYCOB primer amplified a 0.9-kb segment from the DNAs of all 14 species. The MAV and MIN primers each amplified one highly specific 1.3-kb segment from the homologous DNA, respectively. DNAs from each of 10 clinical isolates of M. avium and M. intracellulare identified by conventional methods were amplified with the MYCOB as well as the MAV and MIN primers; 9 of 10 isolates of each species were identified with their respective primers. One isolate of M. intracellulare was subsequently found to have been mislabeled. One isolate designated M. avium reacted only with the MYCOB primer. The hypervariable region of this strain was shown by DNA sequence analysis to be distinct from all known 16S rRNA sequences of Mycobacterium spp. Our data indicate that the currently identified M. avium-M. intracellulare complex includes strains genetically diverse from M. avium and M. intracellulare.