25-Hydroxyvitamin D-binding capacity and affinity were studied in human cord, adult, and maternal sera, and in sera from women receiving oral contraceptives, by in vitro saturation analyses employing dextran-coated charcoal to adsorb unbound sterol. 25-Hydroxyergocalciferol and 25-hydroxycholecalciferol were equipotent in their ability to displace 3H 25-hydroxycholecalciferol from human serum binding sites. At 0 C, the apparent dissociation constant for the serum binding of 25-hydroxycholecalciferol was low (Kd = 8 × 10−10M). Cord and adult sera had a similar 25-hydroxycholecalciferol binding capacity (1.8 × 10−6), but the binding capacity of maternal sera and the sera from women receiving oral contraceptives was significantly higher. At physiological serum concentrations of 25-hydroxyvitamin D (5 × 10−8), only 2–3% of human serum 25-hydroxyvitamin D-binding sites are occupied.