The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3) on calcium transport was studied in vascularly perfused duodena of normal, vitamin D-replete chicks. Addition of 130 pM 1,25(OH)2D3 to the perfusate resulted in a significant increase in 45Ca transport from the lumen to the vascular effluent within 14 min; the transport rate rose to 140% of levels in comparable preparations exposed for 40 min to vehicle. No effects of 1,25(OH)2D3 were noted on the back flux or transfer of 45Ca from the vascular effluent to the lumen. Vascular perfusion with 100 microM colchicine, an antimicrotubular agent, abolished the rapid lumen-to-vascular effluent effect of 1,25(OH)2D3 on 45Ca transport, relative to preparations exposed to the secosteroid and 100 microM lumicolchicine, (a light inactivated analog of colchicine). Colchicine did not, however, alter basal 45Ca transport rates. Addition of 130 pM 1,25(OH)2D3 to the lumenal compartment of normal chicks or vascular perfusion of duodena from vitamin D-deficient birds failed to increase 45Ca transport above control levels. Perfusion of duodena from normal chicks with 650 pM 1,25(OH)2D3 further increased calcium transport to 170% of levels observed in preparations treated with 130 pM steroid, and 210% of levels in controls. Although 15 nM vitamin D3 had no effect, in one series of experiments 125 nM 25-hydroxyvitamin D3 elicited vascular calcium levels that were 185% of controls at 40 min. These results suggest that 1,25(OH)2D3 can act in vitamin D-replete animals to produce rapid unidirectional calcium transport responses (through unknown mechanisms), as well as by interaction with intestinal nuclear receptors in D-deficient animals to promote induction of protein(s) that support long acting calcium transport responses.