N′-Nitrosonornicotine (NNN) is believed to play an important role as a cause of cancer in people who use tobacco products and is considered to be a human carcinogen. NNN requires metabolism to form DNA adducts, which are absolutely critical to its carcinogenic properties. Previous studies have identified cytochrome P450-catalyzed 2′- and 5′-hydroxylation of NNN as potential DNA adduct forming metabolic pathways. 5′-Hydroxylation is the more prevalent of these in monkeys and humans and is known to generate mutagenic intermediates, but the DNA adducts formed by this pathway have never been characterized. In this study, we used 5′-acetoxyNNN as a stable precursor to 5′-hydroxyNNN and investigated its esterase-catalyzed reactions with deoxyguanosine (dGuo) and DNA. Adducts resulting from carbocation and oxonium ion intermediates, produced by the spontaneous decomposition of 5′-hydroxyNNN, were identified. The carbocation pathway resulted in the formation of 2-[2-hydroxy-5-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (12) which was characterized by comparison to an independently synthesized standard. Treatment of 12 with NaBH3CN produced two diastereomers of 2-[2-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (14), and their absolute configurations at the 2-position were determined by comparison to synthetic standards. The oxonium ion pathway produced diastereomers of N 2[5-(3-pyridyl)tetrahydrofuran-2-yl]dGuo (16), identified by comparison to synthetic standards. The absolute configuration at the 5-position was determined by establishing the stereochemistry of the enantiomers of 5-(3-pyridyl)-2-hydroxytetrahydrofuran at the 5-position and allowing these to react individually with dGuo. Treatment of 16 with NaBH3CN produced N2[4-hydroxy-4-(3-pyridyl)but-1-yl]dGuo (18) which was also synthesized independently. Using liquid chromatography-electrospray ionization-tandem mass spectrometry with selected reaction monitoring, we identified adducts 12 and 16 as products of the reactions of 5′-acetoxyNNN with dGuo, Similarly, adducts 14 and 18 were identified as products of the reaction of 5′-acetoxyNNN with DNA followed by NaBH3CN treatment and enzymatic hydrolysis. These results provide the first structural characterization of DNA adducts that can be formed by 5′-hydroxylation of NNN.