A vitamin D-dependent calcium-binding activity of relatively high molecular weight has been identified in the particulate fraction of rat small intestinal mucosa. The Ca-binding activity is sedimented at 140,000 X g after treatment of the mucosal particulate fraction with Triton X-114. Intestinal brush-border suspensions can also be used as starting material. The Ca-binding component is inactivated by heat and repeated freeze-thawing and consists of one or more protein complexes in the range of 0.5-1.0 million mol wt as indicated by gel filtration. The Ca-binding activity correlates positively with known features of the intestinal Ca transport mechanism, as demonstrated by studies of the distribution in the small intestine and the effects of vitamin D, dietary Ca, cycloheximide treatment, and rat age. It is suggested that the component might function in the transit of Ca across the brush-border surface to the cytosol of intestinal mucosal cells.