High field31 P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P 2 O 7 4؊ ) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 ؎ 25.9 and 37.6 ؎ 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 ؎ 17.0 and 36.0 ؎ 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with 32 P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by 31 P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.Infections caused by Trypanosoma cruzi are among the most widespread parasitic diseases in Latin America and are responsible for heavy socioeconomic losses. There is therefore considerable interest in developing novel chemotherapeutic approaches, based on unique aspects of the structure and metabolism of this parasite. T. cruzi develops intracellularly in its vertebrate hosts and is confronted during its life cycle with drastic changes in its microenvironment. Survival through such complex environmental changes requires appropriate reserves of carbon and energy sources, as well as signaling species, such as Ca 2ϩ . Recent work has led to the identification of an acidic calcium pool (acidocalcisome) in this organism (1-4) that contains most of its cellular Ca 2ϩ , together with large amounts of magnesium, sodium, zinc, and phosphorus (3). However, the precise chemical nature of the phosphorus compound(s) in these vacuoles was not determined.In this work, we used 31 P nuclear magnetic resonance (NMR) spectroscopy to investigate the nature of the acidocalcisomal phosphorus.31 P NMR has been extensively used in the past to study the energy metabolism of several microorganisms, as well as that of living vertebrate tissues (5, 6). It has been particularly useful in the identification of phosphorus-based storage compounds, such as polyphosphates (7,8), and sugar phosphates and diphosphates (9, 10). Our results indicate that pyrophosphate is more abundant than ATP in the replicating forms of the parasite and is partly located in acidocalci...