Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and N′-nitrosonornicotine (NNN, 2) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2′-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2′-deoxycytidine and 2′-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2′-deoxycytidine adduct, identified as its stable cytosine analogue O2-[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine (12), was O2-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (13), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (14) and N4-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxycytidine (15) were also observed. The potential conversion of relatively unstable 2′-deoxycytidine adducts to stable 2′-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2, and demonstrate the presence of previously unidentified 2′-deoxycytidine adducts in this DNA.