A Ca2+ indicator arsenazo III was used to demonstrate calcium uptake activity of symbiosomes and the peribacteroid membrane (PBM) vesicles isolated from broad bean root nodules and placed in the medium containing ATP and Mg2+ ions. This process was shown to be rapidly stopped by vanadate, completely reversed in the presence of the calcium ionophore A23187 but insensitive to agents abolishing electrical potential or pH difference across the PBM. The presence of an endogenous calcium pool within isolated symbiosomes and bacteroids was detected using a Ca2+ indicator chlortetracycline. These results prove a primary active transport of Ca2+ through the PBM of legume root nodules and provide the first functional identification of an ATP-driven Ca2+-pump, most likely Mg2+-dependent Ca2+-translocating ATPase, in this membrane.