Abstract In the present work, we discuss the way in which the parallel application of the patch-clamp technique and the 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence detection for recording luminal proton changes allows the functional characterization of nonelectrogenic potassium/proton vacuolar antiporters of the NHX (Na⁺/H⁺ exchanger) family. Moreover, we review the functional role of the tonoplast-specific phosphoinositide PI(3,5)P₂, able to simultaneously inhibit the activity of NHXs and CLC-a transporters, whose coordinated action can play an important role in the water balance of plant cells.