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N(5)-Substituted formamidopyrimidine adducts have been observed from the reaction of dGuo or DNA with aziridine containing electrophiles, including nitrogen mustards. However, the role of substituted Fapy-dGuo adducts in the biological response to nitrogen mustards and related species has not been extensively explored. We have developed chemistry for the site-specific synthesis of oligonucleotides containing an N(5)-nitrogen mustard Fapy-dGuo using the phosphoramidite approach. The lesion was found to be a good substrate for Escherichia coli endonuclease IV and formamidopyrimidine glycosylase.