Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2′-position of the tobacco specific carcinogen N′-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH2OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH2OAc) with DNA, dCyd, and dThd. NNKCH2OAc and NNALCH2OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH2OAc and NNALCH2OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 °C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH2OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH2OAc were similarly characterized. The dThd adduct of NNKCH2OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH4 gave material, which was identical to that produced upon reaction of NNALCH 2OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH2OAc with DNA are O2[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O 2[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH2OAc are O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) and O2[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH2OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH2OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH2OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.