The energy derived from pyrophosphate (PP i ) hydrolysis is used to pump protons across the tonoplast membrane, thus forming a proton gradient. In a plant's cytosol, the concentration of PP i varies between 10 and 800 m, and the PP i concentration needed for one-half maximal activity of the maize (Zea mays) root tonoplast H ϩ -pyrophosphatase is 30 m. In this report, we show that the H ϩ -pyrophosphatase of maize root vacuoles is able to hydrolyze PP i (Reaction 2) formed by Reaction 1, which is catalyzed by PP i -dependent phosphofructokinase (PFP):Fructose-1,6-bisphosphate (F1,6BP) ϩ P i 7 PP i ϩFructose-6-phosphate (F6 P) (reaction 1)During the steady state, one-half of the inorganic phosphate released (Reaction 4) is ultimately derived from F1,6BP, whereas PFP continuously regenerates the pyrophosphate (PP i ) hydrolyzed. A proton gradient (⌬pH) can be built up in tonoplast vesicles using PFP as a PP i -regenerating system. The ⌬ pH formed by the H ϩ -pyrophosphatase can be dissipated by addition of 20 mm F6P, which drives Reaction 1 to the left and decreases the PP i available for the H ϩ -pyrophosphatase. The maximal ⌬ pH attained by the pyrophosphatase coupled to the PFP reaction can be maintained by PFP activities far below those found in higher plants tissues.