Colon cancer is one of the most common cancers in North America and generally develops from colonic epithelial cells following initiation by carcinogens. We have shown that the phase II detoxifying enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1) contributes to the inhibition of carcinogen-induced colon cancer in rats at both the initiation and postinitiation stages. An inactivating polymorphism at base 609 of the NQO1 gene, (609)C (NQO1 *1) --> (609)T (NQO1 *2), occurs at high frequency in the human population. Thus, we carried out a case-control study to determine if this polymorphism is associated with an increased risk of developing colon cancer. A total of 298 patients with colon cancer and 349 healthy controls matched for age, gender, and ethnic origin were enrolled in the study. There was an increased incidence of the NQO1 *2/*2 genotype in patients with colon cancer, with a gender and age-adjusted odds ratio of 2.68 (95% confidence intervals, 1.14-6.28). However, the incidence of the NQO1 *1/*2 genotype was not increased in patients with colon cancer compared with controls. When the patient and control groups were stratified by tobacco and alcohol use, the incidences of the NQO1 *2/*2 genotype were increased in patients with colon cancer for tobacco and alcohol users and nonusers, suggesting that there is no interaction between the NQO1 base 609 polymorphism and tobacco or alcohol use. These results strongly suggest that NQO1 plays a significant role in preventing the development of colon cancer, and individuals with an NQO1 *2/*2 genotype are at an increased risk of developing this disease.