The purpose of this study was to investigate exposure to tobacco smoke related constituents based on the concentration of metabolites in 'spot' urine samples. A total of 200 healthy subjects were recruited over a 9 month period by a CRO from a Canadian phase 1 clinical trial. Subjects were selected based on smoking status, gender and ISO tar yield resulting in 20 male and 20 female non smokers and 40 males and 40 females in each of the tar categories of ≤9 mg and >9 mg. In addition to tar yield, questionnaire data included age, cigarette brand, number of cigarettes smoked, and years of smoking. Urine samples were collected and shipped frozen to the laboratory where, nicotine equivalents, levels of creatinine, TSNA's, PAH's, and arylamines were determined. In comparison with non smokers, smokers had increased (median) urinary concentrations of o-toluidine (196 vs. 178 ng/mg creatinine), 1-aminonaphthalene (62 vs. 29 ng/mg creatinine), 2-aminonaphthalene (20 vs. 14 ng/mg creatinine), and 4-aminobiphenyl (17 vs. 9.9 ng/mg creatinine). With respect to PAHs, non smokers in comparison with smokers had lower median urinary concentrations of hydroxynaphthalenes ( 6.3 vs. 17 ng/mg creatinine), hydroxyphenanthrenes ( 0.26 vs. 0.61 ng/mg creatinine), 2-hydroxyfluorene (1.2 vs. 2.1 ng/mg creatinine), and 1-hydroxypyrene (0.08 vs. 0.25 ng/mg creatinine). Considering TSNAs, the results for smokers were consistent with those reported by others. For each of the biomarkers in this study, notable differences between "≤9 mg" and ">9 mg" tar categories for both male and female subjects were not apparent.