The major polar metabolite of cholecalciferol (vitamin D(3)) present in chick intestinal mucosa has been chemically characterized by mass spectrometric analysis to have a molecular formula of C(27)H(44)0(3) and a structure of 1,25-dihydroxycholecalciferol. This compound, which is produced in the kidney from 25-hydroxycholecalciferol, has been previously shown to be from 4 to 13 times as active as cholecalciferol in stimulating intestinal calcium transport. 1,25-Dihydroxycholecalciferol (previously designated metabolite 4B in this (laboratory) probably represents the biologically active form of cholecalciferol in the intestine.