PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rif r region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens.The genus Mycobacterium comprises more than 70 species, some of which are pathogenic or potentially pathogenic to humans and animals, and some of which are saprobes. Human infections are mainly caused by slowly growing mycobacteria such as Mycobacterium tuberculosis, M. avium complex (MAC), and M. kansasii. Recently, reports of infections due to mycobacteria other than M. tuberculosis complex (MOTT) have been increasing. Because of their clinical importance, in terms of strategies for treatment and the epidemiological implications, the rapid differentiation of causative mycobacteria is important. However, isolation and identification procedures usually require several weeks.Various PCR-mediated methods had been applied for the rapid detection and identification (or differentiation) of mycobacterial species. Among these different methods PCR-restriction analysis (PRA) is preferred, as a simple and cost-effective method that does not involve radioisotopes. It has been applied to several genes, such as 16S ribosomal DNA (rDNA) (6, 9, 10, 33), hsp65 (2, 4, 8, 18, 19, 22, 28, 30, 31), and dnaJ (29), for the rapid differentiation of closely related clinical isolates, such as Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis (8), and subspecies of M. kansasii (18). Previously we reported that comparative sequence analysis of the RNA polymerase gene (rpoB) DNA (342 bp), encompassing the region related to rifampin resistance (Rif r ) in M. tuberculosis, is useful for the identification of mycobacterial...