Three new rif-r-mutations, obtained independently, were localized in the rpoB gene coding for the beta-subunit of DNA-dependent RNA polymerase of E. coli. Two of them led to identical Asp(516)-Asn amino acid substitution with relatively low resistance of corresponding E. coli strains to rifampicin. The third mutation affected the His 526 residue transforming it into Tyr and endowed the E. coli cells with a high resistance against rifampicin.