The role of the calcium pump in the stimulation of intestinal calcium transport activity by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined in chicks. The in situ intestinal absorption of calcium increased approximately threefold in the duodenum, jejunum, and ileum 6 h after a single injection of 625 ng of 1 alpha,25(OH)2D3 into vitamin D-deficient chicks. The same treatment also increased approximately twofold the rate of ATP-dependent calcium uptake by the basolateral membrane vesicles (BL) isolated from those three sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mg(2+)-dependent calcium-stimulated phosphorylated intermediate with an apparent molecular mass of 105 kDa appeared in the BL. The 1 alpha,25(OH)2D3 treatment gave no change in the levels of the intermediate. Pretreatment of the BL with alkaline phosphatase decreased the calcium uptake by the BL isolated from 1 alpha,25(OH)2D3-treated chicks, but it had little effect on the uptake by the BL from vitamin D-deficient chicks. These results suggest that at an early stage of the 1 alpha,25(OH)2D3-induced intestinal calcium transport process, the vitamin regulates the calcium-pumping activity of chick intestinal BL by phosphorylation and dephosphorylation but not by a stoichiometric change in the pump.