Vitamin D and its hydroxylated metabolites are transported in the blood, bound to a transport protein. For this purpose, cartilaginous fish and amphibia use lipoproteins but bony fish, reptiles, birds and mammals have a specific vitamin D-binding protein, called DBP. We have isolated DBP from the serum of man, rat and chick and compared their characteristics. The concentration of DBP is high in the three species (6 to 13 μM) so that less than 3% of all binding sites are normally occupied. The regulation of the serum concentration is quite dissimilar in the three species. There is little age-dependent variation in man and chick but an important increase in serum DBP in the growing rat. Estrogens increase the DBP level in man (and probably also the chick) whereas in the rat androgens increase the DBP concentration. The vitamin D status does not influence the DBP level in any of the species. Human and rat DBP are genetically heterogenous, coded by a pair of codominant autosomes. Human DBP was therefore previously known as group-specific component or Gc. The significance of the serum transport protein for vitamin D can probably be searched in its high affinity and high capacity for 25-hydroxyvitamin D. DBP therefore functions as a storage protein accumulating 25-hydroxyvitamin D during periods of access to vitamin D and releases it, thereafter, very slowly for biological activation or inactivation in the kidney. DBP is also very important in the placental transfer of 25-hydroxy-vitamin D: since the human neonatal DBP level is only half that of the mother, the neonatus also has only half the circulating reserve of 25-hydroxyvitamin D. The DBP level remains constant in most diseases, except in cirrhosis of the liver and nephrotic syndrome. Hypovitaminosis D occurs in the latter disease due to the continuous loss of 25-hydroxyvitamin D together with the loss of DBP in the urine. Serum DBP also interacts with an ubiquitous intracellular protein to form the 6S intracellular binding protein but the significance of this complexation remains to be explored,Finally, DBP is also used in the competitive protein binding assay to measure 25-hydroxyvitamin D in biological fluids. This assay is especially useful to detect (latent) hypovitaminosis D and vitamin D intoxication.