Abstract An investigation of the metabolism of vitamin D 3 and of its biologically active forms in rat kidney and intestinal mucosa was carried out utilizing radiochemically pure H 3-vitamin D 3. The radioactivity present in these tissues 24 hours after the administration of a 500 IU dose of H 3-vitamin D 3 was extracted by the methanol-chloroform procedure of Bligh and Dyer. Forty-nine % of the radioactivity present in intestinal mucosa and 17% of that in kidney was found in the aqueous-methanol phase. The remainder was found in the chloroform layer. Chromatography of the chloroform extract on silicic acid columns and thin-layer plates revealed that a total of 70% of the kidney and 42% of the intestinal radioactivity was present in a material which migrated identically with crystalline vitamin D 3 and which possessed equivalent biological activity when assayed in the rat. At least three other chloroform-soluble radioactive compounds were detected, all of which showed partial vitamin D activity. The aqueous-methanol-soluble radioactive compound(s) were totally without vitamin D activity.