There are around 4000 chemical compounds to which a smoker is exposed when smoking a cigarette. Due to the large variation in individual smoking behaviour, cigarette smoke constituent (CSC) yields are not an accurate representation of uptake by smokers. A better indication of CSC uptake can be obtained by measuring biomarkers of exposure (BoExp) in body fluids. A European, multi-centre, observational study was performed to determine the exposure of adult smokers to selected potentially harmful CSCs. Non-smokers were also included to provide information about background levels of these BoExp. This study was conducted in three countries (United Kingdom [UK], Germany, and Switzerland) according to the principles of good clinical practice. This study included healthy adults in four parallel groups: three groups of smokers stratified according to the tar level of their usual cigarettes (≤4.9, 5.0-7.9, and ≥8.0 mg tar) and one group of non-smokers. Blood and urine samples were taken at two visits and analysed for BoExp for 10 CSCs using validated methods. BoExp levels in smokers and non-smokers were compared using the Wilcoxon rank sum test. Differences between the BoExp levels for the three smoking groups were tested using analysis of variance with adjustment for confounders, followed by pair-wise comparisons. A total of 1250 smokers and 417 non-smokers were enrolled and data from 1159 of the smokers and 399 of the non-smokers were evaluated. Smokers were found to have significantly higher levels of all BoExp compared to non-smokers. There was an association found between BoExp levels and both daily cigarette consumption and tar levels. The highest levels of BoExp were observed in smokers of the highest tar level cigarettes and in smokers with the highest cigarette consumption. This must not, however, be taken as an indication that the risk of smoking low tar cigarettes is any less than that of smoking cigarettes with a higher tar yield. This study provides a snapshot of exposure to a panel of potentially harmful CSCs in three European countries and provides important information on some factors associated with exposure levels.