A new Escherichia coli RNA polymerase mutant was isolated which exhibited reduced accuracy of chain elongation in vivo and in vitro. The novel isolation procedure consisted of simultaneous selection for rifampicin resistance and screening for increased leakiness of an early, strongly polar nonsense mutation of lacZ, one of a special class of mutations whose leakiness reflects mainly transcriptional rather than translational errors. The spontaneous mutant thus isolated displayed a 3-4-fold increase in the leakiness of two different lacZ mutations of this class. Transduction analysis indicated that a single mutation, mapping in or very near the rpoB gene for the beta subunit of RNA polymerase, conferred both rifampicin resistance and increased nonsense leakiness. In an in vitro fidelity assay, homogeneous RNA polymerases from the mutant and parent strains exhibited error rates of 1/0.90 X 10(5) and 1/2.0 X 10(5), respectively, for the poly[d(A-T)] X poly[d(A-T)]-directed misincorporation of noncomplementary GMP. These error rates were verified by product analyses which further revealed that GMP was misincorporated in place of AMP in the synthesis of poly[r(A-U)]. The error rate of wild-type K12 RNA polymerase from a different source was 1/2.0 X 10(5), while that of a hybrid RNA polymerase, containing mutant core enzyme and wild-type sigma subunit, was 1/0.64 X 10(5). These error rates confirmed the selection of a transcriptional accuracy mutant. The error frequencies observed are much lower than those reported in other in vitro assays. The safeguards used to avoid artifactually enhanced misincorporation, and to thereby quantitate lower error rates, are discussed.