Toxoplasma gondii tachyzoites were fractionated by modification of an iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, X-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in phosphorus, calcium, and magnesium, as found before for acidocalcisomes. Staining with 4’,6-diamidino-2-phenylindole (DAPI) indicated that polyphosphate (polyP) was preferentially localized in this fraction together with pyrophosphate (PPi). Using an enzyme-based method, millimolar levels (in terms of Pi residues) of polyP chains of less than 50 residues long and micromolar levels in polyP chains of about 700-800 residues long, were found to be preferentially localized in this fraction. The fraction also contained the pyrophosphatase and polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using antibodies against proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii polyP levels rapidly decreased upon exposure of the parasites to a calcium ionophore (ionomycin), to an inhibitor of the V-H+-ATPase (bafilomycin A1), or to the alkalinizing agent NH4Cl. These changes were in parallel to an increase in intracellular Ca2+ concentration, suggesting a close association between polyP hydrolysis and Ca2+ release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of polyP metabolism in response to cellular stress