Calbindin-D9k, a vitamin D-dependent Ca2+-binding protein, is closely associated with the transcellular absorption of calcium by mammalian enterocytes. Studies were performed to determine whether physiological concentrations of calbindin-D9k altered Ca2+ transport by the ATP-dependent Ca2+ pump in rat duodenal basolateral membrane vesicles. In solutions where free Ca2+ was buffered by EGTA, only a small stimulation of Ca2+ uptake rates could be demonstrated, and it was likely that this was secondary to changes in free Ca2+ concentration. However, a threefold stimulation of uptake by 30 microM calbindin-D9k was found when EGTA-free solutions were used, and changes in free Ca2+ activity or 45Ca2+ specific activity were avoided. The affinity for Ca2+ was reduced in this system but appeared to be stimulated by either calbindin-D9k or EGTA. Other Ca2+-binding proteins that bind Ca2+ in the micromolar range were found to increase Ca2+ uptake in the absence of EGTA. These experiments suggest that one of the actions of calbindin-D9k is to stimulate the rate of extrusion of Ca2+ from the enterocyte by increasing Ca2+ transport by the Ca2+ pump.