The primary synthesis of functional vitamin D begins in the skin, where a cholesterol by-product acted upon by UV light forms vitamin D3. Vitamin D3 is carried through the bloodstream, attached to the vitamin D binding protein, to the liver where it is converted to 25-hydroxy vitamin D3 (25(OH)D3). This product is then transported to the kidney where it is further converted to 1,25-dihydroxycholecalciferol (1,25(OH)2D3), the biologically active form. Active 1,25(OH)2D3 is very short-lived and is rapidly metabolized to the deactivated forms 24,25(OH)2D3 and 1,24,25(OH)3D3. Cayman's Vitamin D EIA Kit is a competitive assay that can be used for quantification of Vitamin D in plasma or serum. Because of the short half-life of the biologically active 1,25(OH)D form, this assay primarily detects the more metabolically stable forms, 25(OH)D3 and 25(OH)D2. Accurate detection of these forms requires that they be displaced from the vitamin D binding protein prior to measurement. A simple, acetone-based purification protocol is included as the initial procedure of the kit for this purpose.