Vitamin D-replete (+D) and vitamin D-deficient (-D) rats received by intraperitoneal injection varying amounts of 1,25-dihydroxyvitamin D3, and 4 h (+D) or 9 h (-D) later everted duodenal sacs were prepared to evaluate active calcium transport, i.e., the amount of calcium found in the serosal fluid. At the same time, duodenal calcium-binding protein (CaBP) content was measured. Calcium transport was a close positive function of CaBP content. It was not detectable when CaBP content was zero and increased linearly without plateauing as CaBP content increased to 100 nmol calcium bound/g mucosa. Trifluoperazine (TFP) inhibited active calcium transport in a concentration-dependent manner. Experiments using vesicles prepared from brush-border or basolateral membranes indicated that TFP inhibited the calcium-extrusion process, with virtually no effect on calcium entry. It is concluded that vitamin D exerts its major regulation of active calcium transport in the rat duodenum via CaBP on transport steps beyond brush-border entry.