Objective: The purpose of this investigation was to examine the effect of an acute swimming exercise session on nucleotidase activities in rat blood serum. Methods: Male Wistar rats were divided in exercise and sedentary groups. In the exercise group, rats were submitted to one swimming session (60 minutes) with a constant load of 4% of body weight in the tail. Nucleotidase activities were gauged spectrophotometrically by measuring the inorganic phosphate from ATP, ADP or AMP hydrolysis or p-nitrophenol released from of p-Nph-5’- TMP hydrolysis. Results: The exercise group presented a significant increase in nucleoside triphosphate diphosphohydrolase (55.5% and 43.1%, for ATP and ADP, respectively), 5’- nucleotidase (57.4%), and nucleotide pyrophosphatase/phosphodiesterase (24.2%) activities. Conclusion: Our results have shown that nucleotidases are activated in rat blood serum after one session of aerobic swimming exercise, suggesting that these enzymes may promote an increase in adenosine levels, which might contribute to exercise-mediated vasodilatation.