Abstract Fifteen hours after the intracardiac administration of a physiological dose (0.65 nmole) of radioactive cholecalciferol (vitamin D3) to rachitic chicks, the bulk of the radioactivity in the plasma, liver, and intestine exists as three polar metabolites. These are designated Metabolites 4A, 4B, and 4C on the basis of their chromatographic mobility on silicic acid columns. After doses of 0.65 to 6.5 nmoles of cholecalciferol, the predominant metabolite found in the plasma is 4A with smaller amounts of 4B and 4C. In the intestine, a primary target tissue of cholecalciferol, the predominant metabolite is 4B, with much smaller amounts of 4A and 4C. Metabolite 4A from both the plasma and intestine is tentatively identified as 25-hydroxycholecalciferol on the basis of its migration with 25-hydroxycholecalciferol during silicic acid column chromatography. The biological activity of Metabolites 4A and 4B was examined with an assay which measures intestinal calcium transport, in vivo. Both 4A and 4B have biological activity significantly greater than equivalent amounts of cholecalciferol. The kinetics of the appearance of Metabolite 4B in the intestine and its binding to intestinal chromatin are consistent with the lag in the physiological response to cholecalciferol. When radioactive Metabolite 4A is administered to rachitic chicks, the bulk of the plasma radioactivity remains as 4A, but the predominant metabolite found in the intestine is 4B. These data support the concept that Metabolite 4B may be the biologically active form of cholecalciferol in the intestine and that 25-hydroxycholecalciferol is an intermediate in its formation.