Objective: the objective of this analysis was to compare the levels of biomarkers of exposure (BoExp) to 2-aminonaphthylamine (2-NA), 4-aminobiphenyl (4-ABP), 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), carbon monoxide (CO), nicotine, and pyrene in male and female Japanese smokers of menthol and non-mentholated, cigarettes, and nonsmokers. Methods: post-hoc analysis of data from a multicenter, observational, ambulatory study in male and female Japanese smokers and non-smokers. BoExp were determined in blood for CO (hemoglobin saturation [%COHb]), in plasma for nicotine (plasma cotinine [PCot]), and in 24-h urine for aromatic amines (unmetabolised 2-NA and 4-ABP), nicotine (the molar sum of cotinine, trans-3’-hydroxycotinine, nicotine, and their respective glucuronide conjugates [Neq]), NNK (4-methylnitrosamino)-1-(3-pyridyl)-1-butanol plus glucuronide conjugates [total NNAL]), and pyrene (1-hydroxypyrene plus glucuronide and sulphate conjugates [total 1-OHP]). Nicotine dependence in smokers was assessed using the Fagerström test for nicotine dependence (FTND). Results: the analyzed population consisted of 345 (219 male, 126 female) non-smokers and 648 (417 male, 231 female) smokers including 152 (79 male, 73 female) smokers of menthol cigarettes and 496 (338 male, 158 female) smokers of non-menthol cigarettes. Mean BoExp levels were higher in smokers than in non-smokers: COHb (3.72±1.18 vs. 1.99±0.39 %), PCot (223.61±126.02 vs. 2.50±0.00 ng/ml), Neq (8.04±6.27 vs. 0.34±0.17 mg/day), total NNAL (231.33±194.81 vs. 6.49±7.03 ng/day), total 1-OHP (316.94±238.56 vs. 179.95±575.63 μg/day), 2-NA (19.27±14.78 vs. 3.67±4.05 ng/day), and 4-ABP (12.54±18.07 vs. 3.50±8.48 ng/day). No consistent differences were apparent between menthol vs. Non-menthol smokers: COHb (3.86±1.36 vs. 3.68±1.12 %), PCot (197.70±117.90 vs. 231.54±127.47 ng/ml), Neq (6.84±5.74 vs. 8.41±6.39 mg/day), total NNAL (267.83±249.11 vs. 220.14±173.60 ng/day), total 1-OHP (289.24±208.31 vs. 325.43±246.66 μg/day), 2-NA (17.21±14.51 vs. 19.90±14.82 ng/day), and 4-ABP (11.03±8.89 vs. 13.00±20.05 ng/day). The average FTND score was 4.1±2.1 vs. 4.9±2.0. Trends for increased BoExp levels were generally observed with increasing cigarette tar and nicotine delivery, and number of cigarettes per day (CPD) in smokers of both menthol and non-mentholated cigarettes.