BACKGROUND The objective of this study was to obtain baseline data on biomarkers of exposure (BoE) and biomarkers of potential harm (BoPH) in smokers, former smokers and never-smokers. METHODS This was a cross-sectional study of 80 healthy male and female volunteers over 21 years old, self-selected for smoking status. Subjects were prescreened by medical staff at an independent clinical research unit, within 1 week prior to a single overnight residential visit and sample collection. RESULTS All BoE were able to differentiate between the two smoking groups and smokers from all nonsmokers. There was a strong correlation between cigarettes smoked per day and total urinary nicotine equivalents (TNE; r=0.85). TNE correlated better with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol levels than cigarettes smoked per day (r=0.75 and r=0.56, respectively). Of the BoPH included in this study, seven (11-dehydro-thromboxane B2, 2, 3-dinorthrom-boxane B2, 8-epi prostaglandin F(2alpha), 8-hydroxy-2 deoxyguanosine, cis-thymidine glycol, low-density lipoprotein cholesterol and IgG) were significantly different between the group who smoked more cigarettes per day and never-smokers. These differences became more apparent and extended to the group who smoked 10 or less cigarettes per day, when total urinary recovery values were corrected for creatinine clearance. CONCLUSIONS While BoE clearly differentiate between groups based on self-declared smoking status, most BoPH examined could not do so in a consistent manner. The dynamics of BoPH levels are not well understood. Future studies of BoPH should eliminate potential confounding factors and increase the number of subjects to allow the investigation of genetic polymorphism in metabolic pathways.