The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) both induce nasal tumors in rats and have a common metabolic activation pathway leading to pyridyloxobutylation of DNA. The role of DNA pyridyloxobutylation in rat nasal carcinogenesis has not been evaluated previously. In this study, we used gas chromatography-mass spectrometry to compare levels of 4-hydroxyl-1-(3-pyridyl)-1-butanone-releasing adducts formed by pyridyloxobutylation of rat nasal mucosa DNA after treatment with either NNK, NNN, or deuterated analogues of NNK. The latter were [4,4-D2]NNK, a stronger nasal cavity carcinogen than NNK, and [CD3]NNK, which has carcinogenic activity equivalent to NNK. We also investigated toxicity to the nasal mucosa and levels of O6-methylguanine in the DNA of this tissue in rats treated with NNK and its deuterated analogues. Rats were given three times weekly s.c. injections of the respective nitrosamines for 4 weeks and then sacrificed 24 h after the final injection. The nasal mucosa was separated into the olfactory and respiratory portions. In the rats treated with [4,4-D2]NNK, levels of O6-methylguanine in DNA from both the olfactory and respiratory portions of the nasal mucosa were significantly lower and levels of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts higher than in the rats treated with equivalent doses of the less carcinogenic compounds NNK or [CD3]NNK. 4-Hydroxy-1-(3-pyridyl)-1-butanone-releasing adducts were also detected in the nasal mucosa DNA of the rats treated with NNN. In the comparative study of NNK and its deuterated analogues, the histology of the nasal mucosa did not appear to be markedly different among these groups. Collectively, the results of this study provide strong evidence that DNA pyridyloxobutylation is important in rat nasal cavity carcinogenesis by NNK and NNN.