Abstract Physiological doses of radioactive vitamin D3 are administered to vitamin D-deficient chicks 16 hours prior to the preparation of lipid extracts of small intestine, plasma, kidney, and bone. Analysis of the vitamin D metabolite pattern is carried out via column chromatography on silicic acid, Sephadex LH-20, and Celite (liquid-liquid partition), countercurrent distribution and treatment with periodic acid. The predominant form of the vitamin in the intestine is a polar metabolite (Peak 4Bi) which is homogeneous in all four separation systems, migrates in the area of known dihydroxy-D3-vitamins, loses 46% of its tritium in the course of metabolic formation from [1α, 2α-3H2]vitamin D3 and is insensitive to cleavage by periodate. These data are consistent with the recent identification of Peak 4Bi as 1,25-dihydroxy-vitamin D3 by Holick, Schnoes, and DeLuca (1971) (Proc. Nat. Acad. Sci. U.S.A. 68, 803) and the original proposal by Haussler, Myrtle, and Norman (1968) (J. Biol. Chem. 243, 4055) that this metabolite is the active form of vitamin D in the intestine. The major metabolite present in plasma, kidney, and bone is 25-hydroxy-vitamin D3, but significant amounts of 1,25-dihydroxy-vitamin D3 are detected in all three sites. The plasma dihydroxy-vitamin D3-metabolite profile differs markedly from that of the kidney and bone in that the predominant species is 25, 26-dihydroxy-vitamin D3, with 21,25-dihydroxy-vitamin D3 also being present in measurable quantities. Virtually all dihydroxy-vitamin D3 in kidney and bone exists as the 1,25-dihydroxy derivative. The presence of 1,25-dihydroxy-vitamin D3 in kidney strengthens the proposal that it is produced metabolically from 25-hydroxy-vitamin D3 by a renal enzyme. The association of 1,25-dihydroxy-vitamin D3 with bone and its considerable activity in promoting skeletal dissolution, raises the question as to whether 25-hydroxy-vitamin D3 or the 1,25-dihydroxy-sterol represents the active form directing bone resorption.