Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the β subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369- 25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the β' subunit. The mutation is the Phe793 → Ser substitution, which occurred in an evolutionarily conserved segment of the β' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring α-amanitin resistance. Both streptolydigin and α-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.