Aim: To investigate the effect of tobacco smoke (TS) exposure on the quantity of exhaled carbon monoxide (eCO) and hair nicotine (HN) and to evaluate the relationship between these values. Materials and methods: Included in the study were 96 subjects (64 male, 32 female) divided into 3 groups. The subjects in Group 1 (n = 46) were current smokers, and the subjects in Group 2 (n = 20) and Group 3 (n = 30) were nonsmokers with or without environmental TS exposure, respectively. The eCO level of all of the subjects was measured with a breath CO monitor. Gas chromatography/mass spectrometry were used for quantification of the HN (n = 47). Results: The mean age of the subjects was 39.1 years. The mean levels of eCO were 9.3 ppm, 1.3 ppm, and 1.0 ppm and the mean HN concentrations were 20.9 ng/mg, 2.1 ng/mg, and 0.7 ng/mg in the 3 groups, respectively. There was a significant difference between Group 1 and the other groups according to the levels of eCO and HN concentrations, but the levels of eCO and HN concentrations were similar in Group 2 and Group 3. There was a positive correlation between the levels of eCO and the HN concentrations. The cutoff values of eCO and HN for smokers were 6 ppm and 4 ng/mg, respectively. Conclusion: Although nicotine analysis in some biological samples like hair is specific to TS exposure, these methods are expensive and difficult procedures. Our results suggest that instead of HN analysis, a cheap and easy method like eCO measurement may be used, but further studies with more cases are needed. © TÜBİTAK.