The tobacco-specific nitrosamines N′-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)-DNA adducts. POB-DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB-DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1- butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 12) was the most abundant adduct, followed by O6-[4-(3-pyridyl)-4-oxobut-1-yl]-2′- deoxyguanosine (O6-POB-dGuo, 8), O2-POB-dThd, and O 2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (O2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB-DNA adduct formed in vivo. However, levels of O6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O6-POB-dGuo was more abundant in lung than in liver. O2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB-DNA adducts, support an important role of O6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.