Everted gut-sacs prepared from segments of the proximal small intestine of young rabbits, rats and guinea pigs transport Ca⁴⁵ in vitro from the mucosal to the serosal surfaces against concentration gradients. The active transport mechanism is limited in capacity, is dependent on oxidative phosphorylation, and appears to be relatively specific for Ca⁺⁺ and Mg⁺⁺ in contrast to Sr⁺⁺ and Ba⁺⁺. Vitamin D deprivation in rabbits and rats markedly impairs the capacity for active Ca⁴⁵ transport in vitro. The vitamin thus has an effect directly on the upper small intestine. Neither the active Ca⁴⁵ transport nor the effect of vitamin D on the transport can be explained by an accumulation of citrate and the formation of the calcium-citrate complex.