Highly activated transcription is associated with eukaryotic genome instability, resulting in increased rates of mitotic recombination and mutagenesis. The association between high transcription and genome stability is probably due to a variety of factors including an enhanced accumulation of DNA damage, transcription-associated supercoiling, collision between replication forks and the transcription machinery, and the persistence of RNA-DNA hybrids. In the case of transcription-associated mutagenesis, we previously showed that there is a direct proportionality between the level of transcription and the mutation rate in the yeast Saccharomyces cerevisiae, and that the molecular nature of the mutations is affected by highly activated transcription. Here we show that the accumulation of apurinic/apyrimidinic sites is greatly enhanced in highly transcribed yeast DNA. We further demonstrate that most apurinic/apyrimidinic sites in highly transcribed DNA are derived from the removal of uracil, the presence of which is linked to direct incorporation of dUTP in place of dTTP. These results show an unexpected relationship between transcription and the fidelity of DNA synthesis, and raise intriguing cell biological issues with regard to nucleotide pool compartmentalization.