Using 39 clinical isolates of Mycobacterium strains with a broad range of susceptibility to rifampicin, we examined the relationship between the degree of resistance to rifampicin and mutational sites of the rpoB gene. All rifampicin-resistant strains had missense mutations. Twenty strains (95%) had a mutation in the cluster I region, which has also been reported in Escherichia coli [Jin and Gross (1988) J. Mol. Biol. 202, 45-58], and the remaining one strain had a mutation at codon 381 [Ala-->Val] in the N-terminal region, which has not been reported in E. coli. Among 18 rifampicin-susceptible strains, two had a mutation in the cluster I region and the other three strains had a mutation in the cluster III region. The mutations at codons 513 (5%), 526 (33%) or 531 (43%) in the cluster I region led to high level resistance to rifampicin (50 micrograms ml-1 < or = MIC). The mutations at the other sites, in the cluster III region (codons 679 or 687) and even in the cluster I region (codon 514, 521, or 533), showed low level (MIC = 12.5 micrograms ml-1) or no (MIC < 0.39 microgram ml-1) resistance to rifampicin. These results suggest that mutations in the rpoB gene are, mostly, but not necessarily, associated with rifampicin resistance of M. tuberculosis, and the sites of mutations on the rpoB gene will affect the level of resistance to rifampicin.