AbstractHigh‐levels of transcription through a gene stimulate spontaneous mutation rate, a phenomenon termed transcription‐associated mutation (TAM). While transcriptional effects on specific mutation classes have been identified using forward mutation and frameshift‐reversion assays, little is yet known about transcription‐associated base substitutions in yeast. To address this issue, we developed a new base substitution reversion assay (the lys2‐TAG allele). We report a 22‐fold increase in overall reversion rate in the high‐ relative to the low‐transcription strain (from 2.1‐ to 47‐ × 10−9). While all detectable base substitution types increased in the high‐transcription strain, G→T and G→C transversions increased disproportionately by 58‐ and 52‐fold, respectively. To assess a potential role of DNA damage in the TAM events, we measured mutation rates and spectra in individual strains defective in the repair of specific DNA lesions or null for the error‐prone translesion DNA polymerase zeta (Pol zeta). Results exclude a role of 8‐oxoGuanine, general oxidative damage, or apurinic/apyrimidinic sites in the generation of TAM G→T and G→C transversions. In contrast, the TAM transversions at GC base pairs depend on Pol zeta for occurrence implicating DNA damage, other than oxidative lesions or AP sites, in the TAM mechanism. Results further indicate that transcription‐dependent G→T transversions in yeast differ mechanistically from equivalent events in E. coli reported by others. Given their occurrences in repair‐proficient cells, transcription‐associated G→T and G→C events represent a novel type of transcription‐associated mutagenesis in normal cells with potentially important implications for evolution and genetic disease. Mol. Mutagen. 2013. © 2012 Wiley Periodicals, Inc.