Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca2+, calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg2+ levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg2+ inhibited fast vacuolar (FV) currents with a K1 of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg2+ at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg2+, cytosolic Ca2+ at less than 10 μM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg2+ was present, submicromolar concentrations of cytosolic Ca2+ activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg2+-Ca2+ effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg2+ concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca2+ with an affinity in the submicromolar range and a cytosolic low-affinity Mg2+-Ca2+ binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca2+ and is inhibitory. In conclusion, cytosolic Mg2+ sensitizes SV channels to physiological cytosolic Ca2+ elevations. Furthermore, we propose that cytosolic and vacuolar Mg2+ concentrations ensure that FV channels do not function as a continuous vacuolar K+ leak, which would prohibit stomatal opening.