BEFORE the molecule of vitamin D acts in the control of calcium metabolism it is converted to a functional form in at least two metabolic steps. The first, a side chain hydroxylation at carbon 25, was shown by the elegant experiments of DeLuca and his co-workers^1–4. This, metabolite is synthesized in the liver from vitamin D^5,6 and is the major form of the circulating vitamin in blood plasma, not only of experimental rats¹ and chickens⁷, but also of man⁸. A second more polar metabolite, localized in intestinal cell nuclei^7,9 and having at least three times the biological activity of an equivalent amount of cholecalciferol^10,11, is derived from 25-hydroxycholecalciferol (25-HCC)¹². When it is obtained from chickens that have been given [4-¹⁴C,l-³H] cholecalciferol, there is a loss of tritium from carbon 1 (refs. 7 and 12). The tritium deficiency and the greater polarity are compatible with oxygen insertion at carbon 1 of 25-HCC.