Cigarette smoking during pregnancy has several impacts on fetal development, including teratogenic effects. The objective of this study was to assess whether the toxic substances (cotinine and polycyclic aromatic hydrocarbons) found in pregnant smokers are transmitted to their fetuses. The outcomes were analyzed measuring cotinine and 1-hydroxypyrene in the amniotic fluid and maternal urine, benzopyrene and cotinine in the umbilical cord blood. Through a controlled cross-sectional design, 125 pregnant women were selected and classified according to their smoking status: 37 current smokers, 25 passive smokers and 63 non-smokers (controls). We performed high-performance liquid chromatography to measure substances' concentrations. A post-hoc Tukey's test was used to analyze the differences between the groups. All variables were significantly different between controls and smokers. The mean ratios between the concentration of cotinine in smokers compared to controls were as follows: 5.9 [2.5-13.5], p<0.001 in the urine; 25 [11.9-52.9], p<0.001 in the amniotic fluid; and 2.6 [1.0-6.8], p = 0.044 in the umbilical cord blood. The mean ratios of 1-hydroxypyrene concentration between smokers and controls were 7.3 [1.6-29.6], p = 0.003 in the urine and 1.3 [1.0-1.7], p = 0.012 in the amniotic fluid, and of benzopyrene in umbilical cord blood was 2.9 [1.7-4.7], p<0.001. There were no significant differences between controls and passive smokers. When comparing the three groups together, there were statistical differences between all variables. Thus, the fetuses of pregnant smokers are exposed to toxic and carcinogens substances. To our knowledge, this is the first study to measure 1-hydroxypyrene in the amniotic fluid and benzopyrene in umbilical cord blood by high-performance liquid chromatography when considering pregnant women in relation to smoking exposure only.