Both 25 [25,26-hydrogen-3] hydroxycholecalciferol and [1alpha,2alpha-hydrogen-3] cholecalciferol were added to bovine plasma in vitro. Analysis by gel-filtration and ion exchange chromatography, electrophoresis, ultracentrifugation, competitive binding specificity studies, and plasma stripping showed that vitamin D circulated with a protein of alpha-globulin mobility. This globulin had a much higher affinity for 25-hydroxycholecalciferol while vitamin D3 appeared to be associated first with an alpha-lipoprotein and with time because associated with the alpha-globulin. This alpha-globulin had a molecular weight of approximately 70,000 as determined by gel-filtration. Cholecalciferol appeared to bind tightly to the alpha-lipoprotein and resisted being stripped from the plasma. Thus, alpha-globulin appears to be the major carrier of vitamin D in the blood while the alpha-lipoprotein may aid in the transfer of cholecalciferol from the gut to the liver via the lymph system.