Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5 mM MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 microgram/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na +K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 microgram/ml oliomycin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal lateral membranes possess two distinct mechanisms for Ca-transport: a) ATP-driven Ca-transport and b) Na/Ca-exchange.