Critical to mechanisms of chemical carcinogenesis and the design of chemopreventive strategies is whether procarcinogen bioactivation in an extrahepatic target tissue (e.g., the lung) is essential for tumor formation. This study aims to develop a mouse model capable of revealing the role of pulmonary microsomal cytochrome P450 (P450)-mediated metabolic activation in xenobiotic-induced lung cancer. A novel triple transgenic mouse model, with the NADPH-P450 reductase (Cpr) gene deleted in a lung-specific and doxycycline-inducible fashion (lung-Cpr-null), was generated. CPR, the obligate electron donor for microsomal P450 enzymes, is essential for the bioactivation of many procarcinogens. The lung-Cpr-null mouse was studied to resolve whether pulmonary P450 plays a major role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer by producing carcinogenic metabolites in the target tissue. A liver-Cpr-null mouse was also studied to test whether hepatic P450 contributes predominantly to systemic clearance of NNK, thereby decreasing NNK-induced lung cancer. The numbers of NNK-induced lung tumors were reduced in the lung-Cpr-null mice but were increased in the liver-Cpr-null mice, relative to wild-type control mice. Decreased lung tumor multiplicity in the lung-Cpr-null mice correlated with reduced lung O6-methylguanine adduct levels, without decreases in NNK bioavailability, consistent with decreased NNK bioactivation in the lung. Moreover, lung tumors in lung-Cpr-null mice were positive for CPR expression, indicating that the tumors did not originate from Cpr-null cells. Thus, we have confirmed the essential role of pulmonary P450-mediated metabolic activation in NNK-induced lung cancer, and our mouse models should be applicable to studies on other procarcinogens that require P450-mediated metabolic activation.