The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.