Phenethyl isothiocyanate (PEITC), which occurs in certain cruciferous vegetables, was tested for its ability to inhibit lung tumorigenesis in rats induced by the tobacco-specific nitrosamine 4-(methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in a study involving virtually lifelong administration of both compounds. In addition, two biomarkers of NNK metabolism [4-hydroxy-1-(3-pyridyl)-1-butanone-releasing hemoglobin adducts and 4-(methylnitrosamino-1-3-pyridyl-1-butanol and its glucuronide in urine] were quantified in randomly selected rats during the course of the study. The rats were assigned to groups as follows: NNK, 2 ppm in drinking water, 60 rats; NNK, 2 ppm in drinking water and PEITC, 3 micromol/g NIH-07 diet, 60 rats; PEITC, 3 micromol/g NIH-07 diet, 20 rats; and untreated controls, 20 rats. NNK was added to the drinking water for 111 weeks and PEITC to the diet for 1 prior to NNK administration and then throughout the 111-week course of treatment. There were no significant differences in body weights or survival among the groups. There were no significant effects of PEITC on blood chemistry or hematology. NNK induced lung tumors (adenoma and/or adenocarcinoma) in 70% of the rats. In the group treated with NNK plus PEITC, 5% of the rats had lung tumors, which was not different from that of control rats. PEITC also appeared to inhibit progression of benign to malignant pancreatic tumors. At intervals during the study, blood was withdrawn from selected rats, and 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing hemoglobin adducts, which are formed upon metabolic activation of NNK, were quantified. The hemoglobin adducts were significantly repressed throughout the study in the rats treated with NNK plus PEITC compared to those treated with NNK. The 24-h urine sample of several rats was analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol glucuronide. A 4-6-fold increase in the sum of these metabolites was observed in the rats treated with NNK plus PEITC compared to those treated with NNK. This is also consistent with inhibition of metabolic activation of NNK by PEITC. Collectively, the results of this study provide strong evidence for the efficacy of PEITC as a chemopreventive agent against NNK-induced pulmonary carcinogenesis in rats and indicate that two biomarkers of NNK metabolism, measurable in tobacco consumers, can be modulated in a predictable way by PEITC administration.