Abstract Secondary malignancies are a serious adverse consequence of alkylator chemotherapy. The risk of developing an alkylator-associated malignancy is influenced by genetic background, although the relevant genetic factors are poorly understood. To screen for novel susceptibility factors, we established a mouse model of alkylator-induced malignancy. We exposed mice from 20 inbred strains to the prototypical alkylating agent, N-nitroso-N-ethylurea (ENU). ENU was a potent carcinogen in many of the strains tested, inducing 140 tumors in 240 ENU-treated mice (66% incidence of at least one tumor in evaluable mice), compared with a background incidence of 8% spontaneous tumors in 240 strain-, age-, and sex-matched control mice (relative risk, 8.4; P < 0.0001). A wide variety of tumor histologies were noted, including epithelial carcinomas, soft tissue sarcomas, and hematopoietic tumors. Cancer susceptibility was a heritable trait for the most common tumor types, lung adenocarcinoma (H2 = 0.25), T cell lymphoma (H2 = 0.19), and myeloid malignancies (H2 = 0.10). Quantitative trait locus mapping identified regions on chromosomes 3, 6, 9, and 15 containing candidate genes associated with lung adenoma, lung carcinoma, and lymphoma susceptibility. This novel mouse model recapitulates many features of human alkylator-associated cancer and supports the hypothesis that susceptibility to this syndrome is influenced by inherited polymorphisms that could be used to make informed clinical treatment decisions. (Cancer Res 2006; 66(10): 5029-38)