We have recently proposed that calmodulin (CaM) may mediate calcium transport across the intestinal brush border membrane. Since calcium transport across this membrane varies as a function of cellular location on the villus (the highest rates of transport occur across the brush border membrane from cells near the tip), we tested this hypothesis by determining whether CaM and its principal binding protein in the brush border membrane [a 102,000 mol wt (102K) protein] also showed this gradient of activity along the villus. Cells were sequentially eluted from the tip to the base of the villus, brush border membrane vesicles (BBMV) were prepared from the eluted cells, and CaM, CaM binding, and calcium-accumulating ability were determined for each preparation of BBMV. We observed that BBMV prepared from cells originating near the tip of the villus possessed the greatest calcium-accumulating activity, CaM content, and CaM binding by the 102K protein. All three measurements were reduced in parallel in BBMV prepared from cells originating from more basal regions of the villus. Calcium-accumulating ability correlated with CaM content (r = 0.876) and CaM binding to the 102K protein (r = 0.788); likewise, CaM correlated with CaM binding to the 102K protein (r = 0.928). When 1,25-dihydroxyvitamin D was administered to vitamin D-deficient chicks, the binding of CaM to the 102K CaM-binding protein appeared to increase more rapidly in BBMV from cells near the tip of the villus than in cells from more basal regions, comparable to our previously reported data for 1,25-dihydroxyvitamin D-stimulated calcium accumulation by similarly prepared BBMV. These data support the hypothesis that CaM and the 102K CaM-binding protein are involved in the regulation of calcium flux across the intestinal brush border membrane.