Abstract In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 22.214.171.124) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5′-di- and 5′-triphosphates in an optimum pH range of 6.0–8.0. It is Ca 2+-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P 1, P 5-di (adenosine 5′-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K m (Michaelis Constant) values of 49.2±2.8 and 49.9±10.4 μM and V max (maximum velocity) values of 49.4±7.1 and 48.3±6.9 nmol of inorganic phosphate · min −1 · mg of protein −1 for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.