The ecto-NTP diphosphohydrolase (NTPDase I) activity, insensible to inhibitors of  ATPases and phosphatases, was characterized on the surface of T. cruzi and a 2282 bp cDNA encoding a full-length NTPDase was cloned (Fietto et al., 2004). Trypomastigotes have shown to have a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite's virulence mechanism. To further characterize T. cruzi NTPDase I we performed a heterologous expression of the active recombinant enzyme. In silico analyses of the sequence predicts a possible cleavage signal peptide at amino acid position 36, immediately following an amino-terminal predicted transmembrane segment, thus suggesting that NTPDase I could be produced as a soluble exported protein. Using this information we designed a strategy to express a soluble NTPDase I. Full-lenght NTPDase I cloned in pGEM vector was used as a template to amplify a 1700 bp DNA fragment that was transferred to pET21b vector (that codes for Hexa-HIS at the carboxy terminal of the recombinant fusion protein). This construction was used to transform E. coli BL21 cells. Recombinant protein was expressed after 1 hour of induction. Soluble and insoluble recombinant apyrases were purified using Ni-NTA-agarose and showed specific activity for ATP hydrolysis between 2-17 nmols/mg protein.h^-1. The substrate specificity showed preference for nucleotides tri-phosphate (ATP>GTP>UTP). The activity was higher in presence of Mg²⁺ than Ca²⁺ and the best renaturation pH was 7,5. We concluded that the rNTPDase I was produced in an active form. To evaluated the use of  rNTPDase I in Chagas disease  diagnosis we performed immunoassays  (ELISA) using serum from chagasic dogs as test samples, leishmaniotic dogs as cross-reaction control, uncontaminated dogs as negative control and specific polyclonal anti-serum anti-NTPDase I as positive control. Our results showed positive recognition only with chagasic serum and positive control serum and evidenciate for the first time that these approaches could be used as new diagnosis method. Now we are starting the production of full-rNTPDase I expressed in bacteria to compare biochemical parameters with rNTPDase (without signal peptide) and testing the recombinant recognition with human's chagasic serum.