The plant V‐ATPase is a protein complex of 13 different VHA‐subunits and functions as ATP driven motor that electrogenically translocates H+ into endomembrane compartments. The central rotor extends into the hexameric head that is fixed by peripheral stators to an eccentric membrane domain. The localization and orientation of VHA‐subunits of the head and peripheral stalk region were investigated by in vivo fluorescence resonance energy transfer (FRET). To this end, VHA‐E, VHA‐G, VHA‐H of the peripheral stalks as well as subunits VHA‐A and VHA‐B were C‐terminally fused to cyan (CFP) and yellow fluorescent protein (YFP). Protoplasts transfected with FRET‐pairs of CFP‐donor and YFP‐acceptor fluorophores fused to VHA‐subunits were analysed for FRET by laser scanning microscopy. The result of the C‐termini mapping allows to refine the arrangement and interaction of the subunits within the V‐ATPase complex in vivo. Furthermore, expression of fused VHA‐E and VHA‐H stimulated acidification of protoplast vacuoles, while other constructs had no major effect on vacuolar pH tentatively indicating a regulatory role of these subunits in plants.