The effect of metabolic inhibitors (KCN and 2-deoxy-d-glucose) on the vacuolar proton gradient, monitored by acridine orange, was assayed in Acer pseudoplatanus cells. Potassium cyanide plus 2-deoxy-d-glucose slightly lowered this gradient, while cellular ATP level was strongly decreased and inorganic pyrophosphate (PPi) content was halved. Two phosphatase inhibitors (imidodiphosphate and KF) restored the PPi level in KCN-treated cells, but decreased the vacuolar proton gradient by inhibiting H+-PPiase. These results, hence, suggest that tonoplast H+-PPiase is especially responsible for the maintenance of vacuolar ΔpH and that this enzyme is the major scavenger of cytoplasmic PPi in cells treated with metabolic inhibitors.