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Acidocalcisomes are acidic, calcium storage compartments with a H⁺ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H⁺-pyrophosphatase (H⁺-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H⁺-PPase with pyrophosphate (PP_i) and short and long chain polyphosphates (polyPs) but a lack …