N′-Nitrosonornicotine (NNN) is the most prevalent of the carcinogenic tobacco-specific nitrosamines found in all tobacco products. Previous studies have demonstrated that cytochrome P450-mediated 5′-hydroxylation of NNN is a major metabolic pathway leading to mutagenic products, but to date, DNA adducts formed by this pathway have been only partially characterized, and there have been no studies reported on adducts formed with bases other than dGuo. Because adducts with dAdo and dThd have been identified in the DNA of the livers of rats treated with the structurally related carcinogen N-nitrosopyrrolidine, we investigated dAdo and dThd adduct formation from 5′-acetoxyNNN (3), a stable precursor to 5′-hydroxyNNN (2). Reaction of 3 with dAdo gave diastereomeric products, which were identified by their spectral properties and LC-ESI-MS/MS-SRM analysis as N6-[5-(3-pyridyl)tetrahydrofuran-2-yl] dAdo (9). This adduct was further characterized by NaBH3CN reduction to N6-[4-hydroxy-4-(3-pyridyl)but-1-yl]dAdo (17). A second dAdo adduct was identified, after NaBH3CN treatment, as 6-[2-(3-pyridyl)pyrrolidin-1-yl]purine-2′-deoxyriboside (18). Reaction of 3 with dThd, followed by NaBH3CN reduction, gave O 2-[4-(3-pyridyl)-4-hydroxybut-1-yl]thymidine (11). Adducts 9, 11, 17, and 18 were all identified by LC-ESI-MS/MS-SRM comparison to synthetic standards. The reaction of 3 with calf thymus DNA was men investigated. The DNA was enzymatically hydrolyzed to deoxyribonucleosides, and the resulting mixture was treated with NaBH3CN and analyzed by LC-ESI-MS/MS-SRM. Adducts 11, 17, and 18, as well as the previously identified dGuo adducts, were identified. The results of this study provide a more comprehensive picture of DNA adduct formation by the quantitatively important 5′-hydroxylation pathway of NNN and will facilitate investigation of the presence of these adducts in laboratory animals treated with NNN or in people who use tobacco products.