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.