Methylating agents react with DNA to give a cationic N7-methyl deoxyguanosine adduct, which can undergo hydrolytic opening of the imidazole ring to form 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (Me-FAPy) 1. We developed a four step synthesis of phosphoroamidite 2 in 24% overall yield, which allowed the preparation of oligodeoxynucleotides containing the Me-FAPy lesion. The in vitro replication of the Me-FAPy lesion was examined with eukaryotic and prokaryotic DNA polymerases and the full-length extension products were sequenced by tandem mass spectrometry. The Me-FAPy adduct was found to be mis-coding however, only the initial insertion of dCTP opposite the adduct could be extended.