The vitamin D-induced intestinal calcium-binding protein (CaBP) was quantitated in membranous components of the intestinal mucosa by a specific and sensitive RIA. Inclusion of detergent (Triton X-100) in extraction buffer and in the RIA system was required to release and measure membrane-associated CaBP. Purified brush borders were shown to contain CaBP with a specific activity (micrograms per mg protein) about 12% of that in the total homogenate. By transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose blots electrophoretically, CaBP was immunologically detected in brush borders from vitamin D3-treated chicks, but not in those from vitamin D3-deficient chicks. CaBP was also detected in isolated brush border membrane vesicles by the gel electrophoresis-blot transfer technique. Brush border CaBP was inaccessible to proteolytic hydrolysis by trypsin unless trypsinized in the presence of detergent. CaBP-binding substances were found to be present in purified brush borders, using the gel overlay technique. A specific binding protein with a mol wt in the range of 50,000-70,000 daltons was identified, as well as an avid CaBP binder at less than 14,000 mol wt. These observations provide evidence for the association of a significant fraction of total intestinal CaBP with brush borders in vivo, which might have physiological relevance.