In Mycobacterium tuberculosis, involvement of alterations of the RNA polymerase & beta; subunit in resistance to rifampicin has been described by Telenti et al. To determine if the same correlation could be observed between the mutation of the rpo B gene and clinically isolated M. tuberculosis of the rifampicin-resistant phenotype in Japan, 47 strains of M. tuberculosis of the rifampicin-resistant phenotype, 17 of the rifampicin-susceptible phenotype, and 4 type strains were examined. A 411-base pair (bp) rpo B fragment was amplified by the polymerase chain reaction and subjected to solid phase direct sequencing. By comparing the nucleotides, mutation involving 8 conserved amino acids were identified in 44 of the 47 (93.6%) rifampicin-resistant isolates, but in none of the 17 sensitive isolates and 4 type strains. All mutations found were clustered within a region of 23 amino acids. Thus, similar to the results reported by Telenti et al., substitution of a limited number of highly conserved amino acids encoded by the rpo B gene appears to be the molecular mechanism responsible for resistance to rifampicin in Japanese clinical isolates of M. tuberculosis. Our results suggest that direct DNA sequencing of the rbo B gene may be a reliable method for identifying rifampicin-resistant M. tuberculosis strains among Japanese clinical isolates.