In vitro binding of 25-hydroxycholecalciferol (25-(OH)D3) and 1.alpha.,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the duodenal mucosa cytosol of rachitic rats. Both 25-(OH)D3 and 1,25-(OH)2D3 bind to a macromolecule (sedimentation coefficient 5.5-6 S in low or high ionic strength) with a high affinity (Kd at 4.degree. C = 1.2 .times. 10-9 M and 2 .times. 10-9 M, respectively). Competition and chase experiments indicate that 25-(OH)D3 binding sites differ from that for 1,25-(OH)2D3.