The presence of an Na+/Ca2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na+ gradient-dependent Ca2+ uptake and the inhibition of ATP-dependent Ca2+ accumulation by Na+. The presence of 75 mM Na+ in the uptake solution reduces ATP-dependent Ca2+ transport by 45%, despite the fact that Na+ does not affect Ca2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na+ inhibition of ATP-dependent Ca2+ uptake to 20%, apparently by preventing accumulation of Na+ in the vesicles realized by the Na+-pump.l It was concluded that high intravesicular Na+ competes with Ca2+ from intravesicular Ca2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca2+ transport shows a sigmoidal dependence on the Na+ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na+ is between 15 and 20 mM. Uptake of Ca2+ in the absence of ATP can be enhanced by an Na+ gradient (Na+ inside greater than Na+ outside). This Na+ gradient-dependent Ca2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca2+ of this system is below 1 microM. In contrast to the ATP-dependent Ca2+ transport, there is no significant difference in Na+ gradient-dependent Ca2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca2+ uptake is 5-times greater than the greater than the Na+/Ca2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na+/Ca2+ exchange mechanism is not subject to regulation by 1 alpha, 25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na+/Ca2+ exchange system while it doubles the ATP-driven Ca2+ pump activity.