As a protozoan parasite of hematophagous insects, Trypanosoma rangeli epimastigotes are exposed to reactive oxygen species during development in hosts. In this work, we investigated the role of H(2)O(2) as a modulator of the ecto-phosphatase activity present in living T. rangeli. We observed that H(2)O(2) inhibits ecto-phosphatase activities in the short and long epimastigote forms of T. rangeli. Ecto-phosphatase activity found in the short form was more sensitive than that found in the long form. Moreover, H(2)O(2) inhibited ecto-phosphatase activity of the short form in a dose-dependent manner and this inhibition was reversible after H(2)O(2) removal. This effect was not observed for T. rangeli ecto-ATPase, another ecto-enzyme present on the external surface of T. rangeli. Cysteine, beta-mercaptoethanol, and reduced glutathione were able to revert the enzyme inhibition promoted by H(2)O(2). Catalase and glutathione peroxidase stimulated this ecto-phosphatase activity, whereas superoxide dismutase was not able to modulate this activity. The ecto-phosphatase activity was also activated by FCCP and inhibited by oligomycin. It seems that H(2)O(2) plays a fundamental role in the regulation of cellular processes of these organisms. We showed, for the first time, that these parasites can produce H(2)O(2), and it is able to regulate ecto-phosphatase activity.