Abstract A protein, that binds calcium and is induced by vitamin D 3, has been identified in chick duodenal mucosa. This calcium-binding protein (CaBP) has been separated from other proteins in the mucosal supernatant fraction and partially purified (30-fold), using dextran gel filtration (Sephadex G-100). Also, CaBP was shown to occur as a distinct electrophoretic band on acrylamide gel. CaBP was detected in all segments of the small intestine and kidney of the vitamin D 3-treated rachitic chick but was not detectable by present methods in colon, liver, and muscle. Thus, the soft tissue localization of CaBP corresponds to known physiological sites of vitamin D 3 action. In the small intestine, the concentration of CaBP in the vitamin D 3-repleted chicks decreases in the order: duodenum > jejunum > ileum, which correlates with the relative abilities of these particular segments to absorb Ca. It is suggested that CaBP may be intimately involved in the translocation of Ca across the intestinal epithelium.