<p><i>Trypanosoma cruzi</i> causes American trypanosomiasis or Chagas Disease, a neglected tropical disease with cardiac, digestive and neurological involvement that can be fatal, and for which there are few effective antiparasitic drugs. EF3 is an elongation factor with ATPase activity present in fungi, which are not present in mammalian; it is essential for protein synthesis in these organisms, this molecule is also present in some protist parasites, so the objective of this work was the determination and characterization of nucleotidases associated with polysomes of <i>T. cruzi.</i> The nucleotidase activity of <i>T. cruzi</i> polysomes was studied and compared with that found in human ribosomes. Epimastigotes of <i>T. cruzi</i> were processed by subcellular fractionation techniques, obtaining the fractions: kinetoplasts (K), polysomal (P) and soluble (S100). The ability to hydrolyze ATP in each fraction was determined measuring the inorganic phosphate (Pi) released. The total ATPase activity was distributed between K (11.6%) and P (9.4%), while S100 did not present activity. The highest specific activity was found in K (116 ± 1 nmol/Pi/mg protein), followed by P (83 ± 3 nmol/Pi/mg protein). The preferential substrate of polysomal nucleotidases was ATP, followed by GTP. Ouabain and vanadate inhibited polysomal ATPase activity by 39% and 68%, respectively. Sequence comparison analysis of EF3 and <i>T. cruzi</i> nucleotidases and molecular modeling were performed, demonstrating that nucleotidase activity does not correspond to a possible EF3 analogue in <i>T. cruzi</i>. Differences with human ribosomal ATPase could be exploited for chemotherapeutic control of the parasite.</p>