The aims of this study were (1) to explore the behavioral and sociodemographic factors influencing urinary cotinine (COT-U) levels in active smokers and in environmental tobacco smoke (ETS)-exposed individuals, (2) to assess the specificity and sensitivity of the questionnaire for identifying active smokers and nonsmokers, and (3) to derive the upper reference value of COT-U in non-ETS exposed individuals. The COT-U levels of 495 adults (age range 18–69 years) who classified themselves as active smokers (29%) or as nonsmokers with (17%) or without (83%) ETS exposure were quantified by LC-MS-MS (quantification limit: 0.1 μg/L, range of linearity: 0.1–4000 μg/L). Median COT-U levels in these groups were 883, 1.38, and 0.39 μg/L, respectively. Significant determinants of COT-U levels in active smokers were the number of cigarettes per day, type of smoking product, smoking environment, as well as time between the last cigarette and urine collection. Among ETS-exposed nonsmokers, significant determinants were living with smokers, being exposed to smoke at home, ETS exposure duration, as well as time between the last exposure and urine collection. When a 30-μg/L COT-U cut-off value was used to identify active daily smoking, the sensitivity and specificity of the questionnaire were 94% and 98%, respectively. For ETS exposure, the COT-U value of 1.78 (0.90 confidence interval 1.75–1.78) μg/L, corresponding to the 95th percentiles of the COT-U distribution in non-ETS-exposed participants, is proposed as upper reference value to identify environmental exposure.