We have identified the presence of polyphosphate kinase (PPK) for the first time in Leishmania donovani amastigotes, the causative pathogenic form of leishmaniasis. Digitonin permeabilized L. donovani amastigote cells in presence of short chain polyphosphate and ADP or GDP produced either ATP or GTP, respectively. ATP and GTP were quantified by coupling with the enzymes hexokinase and glucose-6-phosphate dehydrogenase. Maximum PPK activity was observed for wild-type, sodium stibogluconate resistant and paromomycin resistant AG83 L. donovani amastigotes when ADP was the phosphate group acceptor from polyphosphate. This activity was 29 times higher than the PPK activity when GDP was the phosphate group acceptor from polyphosphate. When AMP was the phosphate group acceptor from polyphosphate, the activity of PPK was 97 times lesser than the activity of PPK when ADP was the phosphate group acceptor from polyphosphate. Existence of both PPK1 and PPK2 is probable in L. donovani amastigotes.