In order to determine the plasma level of 25-hydroxyvitamin D3 (25-OH-D3) in Japanese children by the competitive protein binding assay, the binding proteins were studied in vitamin D-deficient rats. By using serum and kidney cytosol of vitamin D-deficient rats, a standard curve was obtained with high sensitivity and accuracy which allowed the determination of the 25-OH-D3 level (ranging from 0.5-5.0 ng). The plasma 25-OH-D3 level was 21.6 .+-. 10.1 ng/ml (n = 17) in healthy infants and children from 1-15 yr old and 11.4 .+-. 8.6 ng/ml (n = 27) in mature neonates up to 2 days after delivery.