4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung-specific tobacco carcinogen. Metabolism is critical to its elimination given its lipophilic nature. Although NNK can be metabolized through detoxification pathways that safely eliminate it from the body, it can also be bioactivated, resulting in the formation of potentially carcinogenic DNA adducts. The isolated perfused rat lung (IPRL) system was used to determine the effect of NNK perfusate concentration (0.1 and 1.2 μM) on the formation and distribution of metabolites, the level of individual DNA adducts, and total covalent binding in the lung. Coadministration of the chemopreventive agent phenethyl isothiocyanate (PEITC; 20 μM) was also examined to determine its effect on NNK metabolism. NNK was readily metabolized in the IPRL system. In the 0.1 μM perfusions approximately 55% of metabolites formed were through detoxification pathways, whereas roughly 30% were the result of bioactivation pathways. An increase in NNK concentration increased the percentage of unmetabolized NNK and decreased the apparent metabolic clearance in the lung, but the metabolite profiles remained similar between concentrations. The addition of PEITC reduced the formation of oxidative metabolites and increased 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol (NNAL) formation and the percentage of unmetabolized NNK. PEITC also significantly decreased the formation of DNA adducts in the lung tissue. The level of O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd) and O6-[4-(3-pyridyl)-4-oxobut-1-yl]- 2′-deoxyguanosine (O6-POB-dGuo) decreased by 70 to 75%, and that of O6-methylguanine (O6-methyl-Gua) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) decreased by 40 to 45%. Pyridylhydroxybutyl-DNA adducts were not detected in any of the treatment groups. Thus, the IPRL system is useful in determining pulmonary metabolism and DNA adduct formation separate from other metabolizing organs.