The molecular dosimetry of O6-methylguanine (O6MG) in DNA from lung and specific cell populations isolated from lung was determined during multiple administrations of the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to Fischer 344 rats. O6MG accumulated with doses of NNK ranging from 0.1 to 100 mg/kg/day. The dose response for NNK was nonlinear; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low and high Km pathways may exist for activation of NNK to a methylating agent. Marked differences in O6MG concentration were observed in specific lung cell populations. The Clara cell, one of the suggested progenitor cells for nitrosamine-induced neoplasia, was found to possess the greatest concentration of O6MG. Moreover, as the dose of NNK was decreased from 100 to 0.3 mg/kg, the alkylation efficiency in this cell population increased 38-fold. The high level of DNA adduct formation in Clara cells following low dose exposure to NNK was supported by autoradiographic studies. Four h after treatment with 1 mg/kg [3H]NNK, silver grains were more heavily concentrated over Clara cells than over other cell types in the lung. Comparative studies on dimethylnitrosamine, a weak carcinogen in the rat lung, did not demonstrate this cell specificity for DNA alkylation. Thus, the presence of a high affinity pathway in the Clara cell for activation of NNK may contribute to the carcinogenicity of this tobacco specific carcinogen.