Exposure to the tobacco-related nitrosamines 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine is carcinogenic to humans. Metabolic activation of NNK leads to the formation of DNA adducts, which play a critical role in NNK carcinogenesis. Adducts specific to NNK result from covalent linkage of a pyridyloxobutyl (POB-1-yl) group to DNA. Furthermore, some such adducts are unstable, releasing the degradation product 4-hydroxy-1-(3-pyridyl)-1-butanone (4-HPB). Previous qualitative reports from our laboratory have established the chemical structures of the major POB-1-yl-DNA adducts. In this study, we have quantitated the levels of each of these adducts in vitro, as well as their contribution to the biomarker of DNA pyridyloxobutylation, 4-HPB. Standards for the POB-DNA adducts O 6-(POB-1-yl)dGuo, 7-(POB-1-yl)Gua, O2-(POB-1-yl)dThd, and O2-(POB-1-yl)Cyt were synthesized and used to determine standard responses by reverse phase HPLC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS). DNA was incubated with varying amounts of 4- (acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone in the presence of an esterase, conditions favorable to the formation of an active pyridyloxobutylating agent. After sequential enzymatic and neutral thermal hydrolysis, isolation, and purification, the pyridyloxobutylated mixture was analyzed by HPLC-ESI-MS/MS to quantify the relative level of each of these four adducts as well as the released 4-HPB. The most abundant product was 4-HPB, which accounted for two-thirds of the analyzed mixture. The highest adduct levels measured were those of bases that result from loss of deoxyribose upon neutral thermal hydrolysis. These adducts, 7-(POB-1-yl)Gua and O 2-(POB-1-yl)Cyt, comprised an average of 23 and 6% of the analyzed mixture, respectively. O2-(POB-1-yl)dThd and the mutagenic adduct O6-(POB-1-yl)dGuo were detected at the lowest levels, 4 and 2%, respectively. The relative levels of adducts determined in this study provide further insight regarding the chemical reactivity of the activated form of NNK with respect to DNA bases. Furthermore, the analytical standards and mass spectrometric methods used lay the groundwork for establishing a representative array of pyridyloxobutylation adducts as biomarkers of tobacco exposure in further biochemical and in vivo studies.