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The aim of this study was to investigate the possible correlation between smoking status and biomarkers of exposure (BoE) and biological effect (BoBE) in monozygotic twins discordant for smoking status (smoker and non-smoker pairs). By eliminating potential genetic variability in this manner, a clearer pattern of the effects of lifestyle and environmental exposures should become apparent.This was a cross-sectional study on monozygotic healthy twins (44 subjects, 26 males and 18 females) with a mean age 31.5 years. BoE to cigarette smoke and BoBE were measured in body fluids (24 h urine and blood) after medical pre-screening.All BoE were significantly higher in the smoker twins. Among BoBE, 11-dehydrothromboxane B2 (11-dehydro TBX), 2,3-dinorthromboxane B2 (2,3-dinor TBX), 8-epi-prostaglandin F2α (8-epiPGF), hydroxyproline (OH-P), fibrinogen, white blood cell (WBC), neutrophil and lymphocyte counts and heart rate were statistically significantly increased in the smoker compared to the non-smoker twins. Moreover, statistically significant correlations between neutrophil count and 11-dehydro TBX (r = 0.32), WBC and 8-epiPGF (r = 0.33), OH-P and 8-epiPGF (r = 0.49) and heart rate and fibrinogen (r = 0.46) were observed.The study results confirmed the reliability of the BoE for the evaluation of smoking status. Moreover, a subset of the BoBE, reported as being associated with inflammatory conditions and early stages of vascular disorders, has emerged as showing a consistent relationship with smoking status from the present and the previous studies. By using monozygotic twin pairs, genetic variability has been excluded as a possible source of variability in this study. These results should assist in the interpretation of other population studies using these biomarkers.