The plant vacuole plays a fundamental role in cell homeostasis. The successful application of patch-clamp technique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters. The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation. In excised patch, the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels. In whole-vacuole, the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette. Both vacuolar NHXs and CLCa (chloride/nitrate antiporter) are inhibited by the phosphoinositide PI(3,5)P, suggesting a coordinated role of these proteins in salt accumulation. Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.