The　tobacco-specific　carcinogens　N＇-nitrosonornicotine　(NNN)　and　4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone　(NNK)　require　metabolic　activation　to　exert　their　carcinogenicity.　NNN　and　NNK　are　metabolized　to　the　same　reactive　diazonium　ions,　which　alkylate　DNA　forming　pyridyloxobutyl　(POB)　DNA　base　and　phosphate　adducts.　We　have　characterized　the　formation　of　both　POB　DNA　base　and　phosphate　adducts　in　NNK-treated　rats　and　the　formation　of　POB　DNA　base　adducts　in　NNN-treated　rats.　However,　POB　DNA　phosphate　adducts　in　NNN-treated　rats　are　still　uncharacterized.　In　this　study,　we　quantified　the　levels　of　POB　DNA　phosphate　adducts　in　tissues　of　rats　chronically　treated　with　(S)-NNN　or　(R)-NNN　for　10,　30,　50,　and　70　weeks　during　a　carcinogenicity　study.　The　highest　amounts　of　POB　DNA　phosphate　adducts　were　observed　in　the　esophagus　of　the　(S)-NNN-treated　rats,　with　a　maximum　level　of　5400　+/-　317　fmol/mg　DNA　at　50　weeks.　The　abundance　of　POB　DNA　phosphate　adducts　in　the　esophagus　was　consistent　with　the　results　of　the　carcinogenicity　study　showing　that　the　esophagus　was　the　primary　site　of　tumor　formation　from　treatment　with　(S)-NNN.　Compared　to　the　(R)-NNN　group,　the　levels　of　POB　DNA　phosphate　adducts　were　higher　in　the　oral　mucosa,　esophagus,　and　liver,　while　lower　in　the　nasal　mucosa　of　the　(S)-NNN-treated　rats.　Among　10　combinations　of　all　isomers　of　POB　DNA　phosphate　adducts,　Ap(POB)C　and　combinations　with　thymidine　predominated　across　all　the　rat　tissues　examined.　In　the　primary　target　tissue,　esophageal　mucosa,　Ap(POB)C　accounted　for　similar　to　20%　of　total　phosphate　adducts　in　the　(S)-NNN　treatment　group　throughout　the　70　weeks,　with　levels　ranging　from　780　+/-　194　to　1010　+/-　700　fmol/mg　DNA.　The　results　of　this　study　showed　that　POB　DNA　phosphate　adducts　were　present　in　high　levels　and　persisted　in　target　tissues　of　rats　chronically　treated　with　(S)-　or　(R)-NNN.　These　results　improve　our　understanding　of　DNA　damage　during　NNN-induced　carcinogenesis.　The　predominant　POB　DNA　phosphate　isomers　observed,　such　as　Ap(POB)C,　may　serve　as　biomarkers　for　monitoring　chronic　exposure　of　tobacco-specific　nitrosamines　in　humans.