Treatment of wild-type vaccinia virus infected cells with the anti-poxviral drug isatin-beta-thiosemicarbazone (IBT) induces the viral postreplicative transcription apparatus to synthesize longer-than-normal mRNAs through an unknown mechanism. Prior studies have shown that virus mutants resistant to or dependent on IBT affect proteins involved in control of viral postreplicative transcription elongation, including G2, J3, and the viral RNA polymerase. Prior studies also suggest that there exist additional unidentified vaccinia genes that influence transcription elongation. The present study was undertaken to target candidate transcription elongation factor genes in an error-prone mutagenesis protocol to determine whether IBT-resistant or -dependent alleles could be isolated in those candidate genes. Mutagenesis of genes in which IBT resistance alleles have previously been isolated, namely A24R (encoding the second largest RNA polymerase subunit, rpo132) and G2R (encoding a positive transcription elongation factor), resulted in isolation of novel IBT resistance and dependence alleles therefore providing proof of principle of the targeted mutagenesis technique. The vaccinia H5 protein has been implicated previously in transcription elongation by virtue of its association with the positive elongation factor G2. Mutagenesis of the vaccinia H5R gene resulted in a novel H5R IBT resistance allele, strongly suggesting that H5 is a positive transcription elongation factor.