The polymerase chain reaction (PCR) and automated DNA sequencing were used to detect a genetic locus, rpoB, associated with rifampin resistance in Mycobacterium tuberculosis (TB) in clinical isolates and directly in clinical specimens. Primers derived from the sequence of a TB rpoB gene fragment were used to amplify DNA from bacterial and mycobacterial isolates. An rpoB-specific PCR product was obtained for five of five TB, seven of eight other mycobacterial species, Nocardia sp., Corynebacterium sp., Streptomyces sp., Actinomyces sp., and Rhodococcus sp., but not for 15 isolates (eight genera) representing usual bacterial flora. Sequence comparison of the amplified rpoB region revealed the occurrence of TB-specific "signature nucleotides" at three positions. PCR yielded amplification products for seven of 16 clinical specimens. Five of the seven contained TB-specific DNA, as well as sequences that predicted rifampin susceptibility in accord with agar dilution results. None of ten specimens that were culture negative for TB yielded TB-specific PCR products. These results with a limited number of clinical specimens demonstrate the feasibility of direct detection by PCR of rifampin-resistant TB in clinical specimens. Such testing may serve as a rapid surrogate test for multidrug-resistant TB in laboratories with PCR and automated sequencing capability.