Vacuolar proton pyrophosphatases (V-H؉ -PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H ؉ -PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H ؉ -PPase gene by using double-stranded RNA interference methodology to produce inducible V-H ؉ -PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. brucei V-H ؉ -PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 ؋ 10 5 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.Intracellular acidic vacuoles containing polyphosphate (polyP), 1 initially called volutin or polyP bodies, have been described in bacteria, algae, yeast, and protozoa (1). In trypanosomatids, these polyP vacuoles were called acidocalcisomes (2) and shown to be electron dense and contain large concentrations of PP i , calcium, magnesium, and other elements (3). Similar organelles have been identified in apicomplexan parasites (4, 5) as well as in the green algae, Chlamydomonas reinhardtii (6) and the slime mold, Dictyostelium discoideum (7). Acidocalcisomes were postulated to play an important role in the regulation of both cytosolic Ca 2ϩ concentration and intracellular pH (pH i ). For example, polyP hydrolysis (8) and activation of the Na ϩ /H ϩ antiporter (9, 10) were postulated to protect the cells against alkaline pH stress and increase the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ). In addition, these organelles possess a vacuolar-type H ϩ -translocating pyrophosphatase (V-H ϩ -PPase) (11), which is an electrogenic proton pump initially discovered in photosynthetic bacteria and plants (12). It has been shown to be associated with the plasma membrane or vacuoles in plants and with the chromatophore membranes of Rhodospirullum rubrum. The biochemical function of this enzyme in plants and unicellular eukaryotes is to couple hydrolysis of the high energy phosphate bond of PP i with H ϩ translocation from the cytosol to acidify the plant vacuole (tonoplast) or the acidocalcisome, respectively (10, 13). Working on isolated acidocalcisomes, Rodrigues et al. (14) demonstrated that the Trypanosoma brucei H ϩ -PPase was able to generate a membrane potential by PP i -dependent proton uptake. Moreover, Scott et al. (11,15) showed tha...