The administration of vita-min D(3) to rachitic chicks induces in intestinal mucosal tissue the formation or elaboration of a calcium-binding factor which is found in the supernatant of the mucosal homogenate. The enhanced binding of Ca by the "vitanmin D" supernatant (in contrast to "rachitic" supernatant) was indicated by a slower rate of diffusion of Ca(45) across a cellophane dialyzing membrane and by a lesser amount of Ca(45) being bound to an ion-exchange resin (Chelex-100) in the presence of vitamiiin D(3) supernatant. The binding activity was only associated with the protein fraction from a Sephadex G-25 column and was destroyed by trypsin digestion. This and other evidence suggest that the soluble factor is a protein. The vitamin D(3)-enhanced duodenal absorption of Ca(47) in rachitic chicks occurred almost simultaneously with the appearance of the vitamin D(3)-induced factor, and there was good correlation between the concentration of binding factor and the rate of absorption of Ca(47).