The human plasma 25-Hydroxyvitamin D_3 (25-OH-D_3) was successfully measured by the competitive protein binding assay using the serum or kidney cytosol derived from the vitamin D deficient rat as a binding protein. The standard 25-OH-D_3 could be assayed proportionally from 0.5 to 5.0 ng. The mean plasma 25-OH-D_3 concentration of healthy infants and children was 21.6±10.1 ng/ml and that of mature newborn infants within 48 hours after delivery was 3.5±1.9 ng/ml.