Abstract The goal of the present work was to determine the impact of N3-methyladenine (3-mA), an important lesion generated by many environmental agents and anticancer drugs, on in vivo DNA replication and in vitro RNA transcription. Due to 3-mA chemical instability, the stable isostere 3-methyl-3-deazaadenine (3-m-c3A) was site specifically positioned into an oligodeoxynucleotide. The oligomer was, then incorporated into a vector system that is rapidly converted to ssDNA inside yeast cells and requires DNA replication opposite the lesion for plasmid clonal selection. For control purposes, an adenine or a stable apurinic/apyrimidinic (AP)-lesion was placed at the same site. The presence of each lesion in the oligonucleotide was confirmed by MALDI-TOF analysis. Plasmids were then transfected into yeast cells. While the AP-site dramatically reduced plasmid replication in all strains, the 3-m-c3A had a slight effect in the rad30 background which significantly increased only in a rev3rad30 background. Considering TLS events opposite 3-m-c3A, the lack of Polη was associated with a substantial increase in AT>GC transitions (p=0.0011), while in the absence of Polζ only events derived from an error free bypass were detected. The 3-m-c3A also did not affect in vitro transcription, while the AP-site was a strong block to T7 RNA progression when located in the transcribed strand. We conclude that, in these experimental systems, 3-m-c3A is efficiently bypassed by replication in vivo and by transcription in vitro.