Vacuolar pyrophosphatase (V-PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V-PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V-PPase activity. Samples from 'Valencia' orange had intermediate V-PPase levels. When assayed at equal V-PPase activity (measured as Pi production), V-PPase was not able to generate a pH gradient (DeltapH) in vesicles from acid lime, despite its capacity to form a DeltapH in the presence of ATP. Vesicles from sweet lime and 'Valencia' orange were able to form similar DeltapHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V-PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V-PPase polypeptide from mung bean recognized V-PPase from sweet lime and 'Valencia' orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton-pumping reactions of V-PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.