Abstract A previous study of energy-independent in vitro Ca 2+ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH) 2D 3 repletion ( R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA 74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology 78, 325–332). In the present study, properties of Ca 2+ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg 2+ and Sr 2+, but not by Na + or K +. Lowering the external [H +] or raising the internal [H +] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H +] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl 2 or SrCl 2 but not CaCl 2, NaCl, or KCl. Vesicles preloaded with K 2SO 4 failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl 2 was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca 2+ uptake maintained at equilibrium. Therefore, the remainder of the Ca 2+ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10 −6 m was found for this binding. The bound Ca 2+ could be rapidly released by external Mg 2+ or Sr 2+, but not Ca 2+, Na +, or K +. Release of bound Ca 2+ could also be induced by raising the [H +] of the external medium. Failure of external Ca 2+ to release bound Ca 2+ suggested that the release induced by external Mg 2+, Sr 2+, or H + was not due to competitive displacement of Ca 2+ from its binding sites. These results indicated that Ca 2+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca 2+ to the vesicle. The effects of H +, Mg 2+, and Sr 2+ on Ca 2+ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.