Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase as well as a cofactor of aromatic amino acid hydroxylases. However, its role in pregnancy is not yet understood. We evaluated the concentrations of BH4 throughout normal pregnancy and puerperium, and compared them with those of non-pregnant women by measuring its oxidation product biopterin. In addition, we also measured 6-pyruvoyl tetrahydropterin synthase (PTPS) activities, the rate-limiting enzyme in synthesizing BH4, in pregnant women at the 30th gestational week and non-pregnant women. Although the urinary biopterin levels did not remarkably change, plasma biopterin levels significantly decreased from the 10th gestational week to the 1st day of postpartum compared with those of non-pregnant women. There was no significant difference in PTPS activities between pregnant and non-pregnant women. However, the proportion of reticulocytes, which have been shown to possess high PTPS activity, is significantly higher in pregnant women than in non-pregnant women. Our results suggest that decreased plasma BH4 levels in pregnancy is caused by impaired PTPS activity.