A dominant lethal mutation in the Escherichia coli rpoD gene, which encodes sigma 70, the promoter recognition subunit of RNA polymerase, was isolated after random mutagenesis. The lethal gene was maintained under control of the lac repressor on a low copy plasmid. An amount of lethal sigma 70 that was nearly equimolar with the chromosomally encoded sigma 70 was sufficient to cause cessation of growth. RNA synthesis per unit cell mass was unaffected, but protein synthesis was inhibited by the mutant sigma 70. The amino acid change (Glu-585 to Gln) was in a region of sigma 70 thought to bind the -35 hexamer of the promoter, and the mutant sigma 70 caused increased expression from promoters with nonconsensus bases in the third position of the -35 hexamer. A null mutation of the fis gene could partially suppress the mutant phenotype. These properties are consistent with those expected of a sigma 70 insensitive to growth rate control of rRNA and tRNA promoters.