Intestinal Ca2+ transport was studied in membrane vesicles isolated from microvillus, Golgi, and lateral-basal membrane preparations. Ca2+ uptake by these vesicles was measured by determination of 45Ca2+ associated with these membranes after collection by micropore filtration. Golgi membranes showed the highest initial rate and equilibration level of Ca2+ uptake. Approximately 90% of this Ca2+ uptake was into an osmotically responsive space, suggesting that what was measured was predominantly Ca2+ translocation. Vitamin D-deficient rats showed a markedly diminished rate of uptake and level of equilibration. These data indicate that a Ca2+-translocating process was associated with Golgi membranes to a greater extent than with surface membranes and that this process was markedly decreased in vitamin D-deficient rats. The results suggest that the Golgi apparatus participates in intestinal Ca2+ absorption.