In order to measure the level of toluene in the blood of fetuses of pregnant rats exposed to toluene, application of headspace-solid phase micro extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was studied. Pregnant rats from gestational day 15 (GD15) to GD19 were subjected to inhalational exposure to toluene for 90 min per day. They were obtained by Cesarean section on the fifth day of exposure (on GD19), and the level of toluene in the blood of mother rats and in fetuses were quantified using 5 µl blood. The levels of toluene in the blood of the fetuses in the groups exposed to toluene at 9 ppm and 90 ppm were 0.07 ± 0.03 µg/ml (n = 17) and 2.0 ± 0.51 µg/ml (n = 16) respectively, which were significantly higher than the blood level of the fetuses in the control group (0.02 ± 0.02 µg/ml, n = 12). Meanwhile, the blood concentrations of mother rats were 0.06 ± 0.06 µg/ml (n = 3, control group), 0.14 ± 0.03 µg/ml (n = 3, 9 ppm exposure group), and 3.5 ± 1.5 µg/ml (n = 4, 90 ppm exposure group), respectively, which were higher than those of the fetuses per unit blood volume.