Tonoplast vesicles isolated from juice cells of mature `Valencia' oranges [Citrus sinensis (L.) Osbeck] showed similar tonoplast-bound vacuolar ATPase (V-ATPase) and inorganic pyrophosphatase (V-PPiase) activity as measured by product formation. Both proton pumps were able to generate a similar pH gradient, although steady-state was reached faster with ATP as substrate. When a ΔpH of 3 units was imposed (vesicle lumen pH of 4.5 and incubation medium of 7.5), tonoplast-bound PPiase was not able to significantly amplify the existing ΔpH. Although not able to function as a H+ pump, V-PPiase effectively synthesized PPi in the presence of inorganic phosphate (Pi). Formation of PPi by V-PPiase was enhanced by ATP but inhibited by NaF, gramicidin, and by antibodies raised against V-PPiase from mung bean [Vigna radiata (L.) R. Wilcz. (Syn. Phaseolus aureus Roxb.)]. Immunological analysis demonstrated an increase in V-PPiase protein with fruit maturity. Data indicate that under in vivo conditions, the V-PPiase of mature orange juice cells acts as a source of inorganic pyrophosphate (PPi) but not as a H+ pump. We propose that synthesis of PPi provides a mechanism for recovery of stored energy in the form of the pH gradient across the vacuole during later stages of development and postharvest storage.