The activity and distribution of the metabolic pathways of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and the structurally related nitrosamine, N'-nitrosonornicotine (NNN) were examined in pulmonary cells from F344 rats in order to investigate the mechanisms by which NNK and NNAL, but not NNN, cause lung tumors. The tritium labeled nitrosamines were incubated with Clara cells, alveolar macrophages, alveolar type II cells, or small cells and metabolites were analyzed by HPLC. O6-Methyl-guanine (O6MG ) formation was also quantified in the cells incubated with NNK. Clara cells metabolized all compounds more extensively than the other cell types. Total α-hydroxylation, carbonyl reduction to NNAL, and pyridine N-oxidation in cells incubated with NNK, as well as concentrations of O6MG in DNA were higher in Clara cells than in other cell types. Carbonyl reduction of NNK predominated over the other metabolic pathways in all cell types. The high activity for α-hydroxylation of NNK in Clara cells is consistent with previous studies which proposed that the cell specificity for O6MG formation and the accumulation of this adduct during low-dose exposure to NNK may stem from the presence of a high affinity pathway in Clara cells for NNK activation. Metabolism of NNAL by α-hydroxylation, and by reconversion to NNK followed by α-hydroxylation were observed. Total α-hydroxylation of NNAL was less extensive than α-hydroxylation of NNK. NNN was metabolized by both the 2'- and 5'-α-hydroxylation pathways. 2'-Hydroxylation of NNN produces the same DNA pyridyloxobutylating agent as does methyl hydroxylation of NNK. However, NNN is not a methylating agent and does not induce lung tumors in rats. Metabolism of NNN by 2'-hydroxylation was, depending on cell type, 41-;85% as extensive as total α-hydroxylation of NNK, indicating that the rates of formation of the DNA pyridyloxobutylating agent were similar from NNN and NNK. The results of this study demonstrate that Clara cells have a high capacity to metabolically activate NNK, NNAL and NNN and provide further support for the hypothesis that DNA methylation of pulmonary cells is important in NNK carcinogenesis.