BACKGROUND: During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts. Transcription factor S-II enhances the excision of these mis-incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro. This mechanism is considered to be transcriptional proof-reading, but its physiological relevance remains unknown. RESULTS: We report that S-II contributes to the maintenance of transcriptional fidelity in vivo. We employed a genetic reporter assay utilizing a mutated lacZ gene from which active beta-galactosidase protein is expressed when mRNA proof-reading is compromised. In S-II-disrupted mutant yeasts, beta-galactosidase activity was ninefold higher than that in wild-type. The S-II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S-II gene. The mutant S-II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity. CONCLUSION: These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation.