Kinetic and molecular properties of the Ca2+/H+ antiporter in the vacuolar membrane of mung bean hypocotyls were examined and compared with Ca2+-ATPase. Ca2+ transport activities of both transporters were assayed separately by the filtration method using vacuolar membrane vesicles and 45Ca2+. Ca2+ uptake in the presence of ATP and bafilomycin A1, namely Ca2+-ATPase, showed a relatively low Vmax (6 nmol.min-1.mg-1 protein) and a low Km for Ca2+. The Ca2+/H+ antiporter activity driven by H+-pyrophosphatase showed a high Vmax (25 nmol.min-1.mg-1) and a relatively high Km for Ca2+. The cDNA for mung bean Ca2+/H+ antiporter (VCAX1) codes for a 444 amino-acid polypeptide. Two peptide-specific antibodies of the antiporter clearly reacted with a 42-kDa protein from vacuolar membranes and a cell lysate from a Escherichia coli transformant in which VCAX1 was expressed. These observations directly demonstrate that a low-affinity, high-capacity Ca2+/H+ antiporter and a high-affinity Ca2+-ATPase coexist in the vacuolar membrane. It is likely that the Ca2+/H+ antiporter removes excess Ca2+ in the cytosol to lower the Ca2+ concentration to micromolar levels after stimuli have increased the cytosolic Ca2+ level, the Ca2+-ATPase then acts to lower the cytosolic Ca2+ level further.