Given that defects in cell cycle control and DNA repair capacity may contribute to tumorigenesis, we hypothesized that patients with lung cancer would be more likely than healthy controls to exhibit deficiencies in cell cycle checkpoints and/or DNA repair capacity as gauged by cellular response to in vitro carcinogen exposure. In an ongoing case-control study of 155 patients with newly diagnosed lung cancer and 153 healthy controls, we used the comet assay to investigate the roles of cell cycle checkpoints and DNA damage/repair capability in lung tumorigenesis. The median gamma-radiation-induced and benzo(a)pyrene diol epoxide-induced Olive tail moments, the comet assay parameter for measuring DNA damage, were significantly higher in the case group (5.31 and 4.22, respectively) than in the control group (4.42 and 2.83, respectively; P < 0.001). Higher tail moments of gamma-radiation and benzo(a)pyrene diol epoxide-induced comets were significantly associated with 2.32- and 4.49-fold elevated risks, respectively, of lung cancer. The median gamma-radiation-induced increases of cells in the S and G(2) phases were significantly lower in cases (22.2% and 12.2%, respectively) than in controls (31.1% and 14.9%, respectively; P < 0.001). Shorter durations of the S and G(2) phases resulted in 4.54- and 1.85-fold increased risks, respectively, of lung cancer. Also observed were joint effects between gamma-radiation-induced increases of S and G(2) phase frequencies and mutagen-induced comets. In addition, we found that in controls, the S phase decreased as tail moment increased. This study is significant because it provides the first molecular epidemiologic evidence linking defects in cell cycle checkpoints and DNA damage/repair capacity to elevated lung cancer risk.