Cholecalciferol (vitamin D3) and its 25-hydroxy metabolite are transported in plasma bound to a specific protein, the binding protein for cholecalciferol and its metabolites (DBP). DBP is identical with the group-specific component (Gc) proteins, which are known to display genetic polymorphism. Studies were conducted to explore whether or not major differences in the transport of cholecalciferol and its biological metabolites might exist among persons with different Gc phenotypes. Detailed quantitative studies were first carried out on the interaction of 25(OH)D3 with DBP in 21 different samples of serum, representing eight different Gc phenotypes. The studies used a filter disc assay method that provided highly reproducible quantitative results with cholecalciferol-related sterols. The Gc phenotypes studied included the three common types (Gc 1-1, 2-1, and 2-2) and several uncommon genetic variants (Gc Ab-Ab, Ab-1, Ab-2, Chip-1, and Chip-2). The binding affinities for 25(OH)D3 observed with these different sera were all fairly similar to each other. More extensive studies were then conducted to compare the binding of four cholecalciferol-related sterols to each of three genetic variants of DBP, by using sera from homozygous persons with the Gc 1-1, Gc 2-2 and Gc Ab-Ab phenotypes. The ligands tested included cholecalciferol, 25(OH)D3, 1,25(OH)2D3, and 24(R) 25(OH)2D3. The affinities of the three genetic types of DBP/Gc protein were found to be similar for each of the four cholecalciferol-related sterols. The apparent association constants for 25(OH)D3 and 24,25(OH)2D3 were similar (approx. 1–2 × 10(8) M-1); lesser affinities were observed for 1,25(OH)2D3 (kA approx. 1 × 10(7) M-1) and for cholecalciferol (kA approx. 3–4 × 10(5) M-1). Thus the common genetic variants of DBP/Gc protein, and the uncommon genetic variants studied here, all appear to have similar binding properties for cholecalciferol and its several metabolites.