Nutritional requirements for vitamin D during pregnancy have been inadequately described, and there are conflicting data on the impact of gestation on vitamin D status. In the present study, we conducted a longitudinal analysis of total and free (unbound) serum 25-hydroxyvitamin D (25(OH) D), vitamin D-binding protein (DBP) and albumin concentrations in a random sample of thirty women from the Screening for Pregnancy Endpoints Ireland pregnancy cohort study at 15, 20, 24, 28, 32, 36 and 40 weeks of gestation and at 2 months postpartum. Concentrations of serum 25(OH) D, DBP and albumin were determined, and free 25(OH) D was calculated from the concentrations of total 25(OH) D, DBP and albumin. Serum albumin concentration decreased during pregnancy (P < 0.001), with a nadir at 36 weeks (P < 0.005), during which the concentration was approximately 80% of the postnatal concentration. Serum DBP concentration increased during pregnancy and at 28 weeks of gestation, which was almost double the postnatal level (P < 0.001). Total and free 25(OH) D concentrations decreased (both P < 0.005) as pregnancy progressed, and both were lowest at 36 weeks of gestation. At 15 weeks, 10 and 63% of the women had serum 25(OH) D concentration,30 and 50 nmol/l, respectively, which increased to 53 and 80% at 36 weeks of gestation. The time course of decreasing concentrations of 25(OH) D during gestation among women recruited during May-July, who delivered between October and November, and among those recruited in August-September, who delivered between February and March, was similar. The lower percentage of free 25(OH) D during pregnancy is mainly due to increased DBP.