This study was aimed to determine normal serum adenosine deaminase (ADA) activity in domestic animals in both sexes and that were considered healthy on the basis of clinical and laboratory examinations. In this study, 262 non-pregnant animals of both sexes, belonging to 5 species [cattle (n = 50), sheep (n = 65), goat (n = 52), dog (n = 55), and cat (n = 40)] were used. Serum total ADA and ADA1 isoenzyme activities were found to be 6.34 +/- 0.31 IU/l and 6.34 +/- 0.31 IU/l in cattle, 7.57 +/- 0.30 IU/l and 7.31 +/- 0.47 IU/l in sheep, 7.52 +/- 0.36 IU/l and 6.44 +/- 0.72 IU/l in goat, 2.56 +/- 0.13 IU/l and 2.03 +/- 0.30 IU/l in dogs, 23.36 +/- 1.59 IU/l and 21.71 +/- 2.13 IU/l in cats, respectively. The serum levels of ADA2 were low in sheep, goats, dogs, and cats, whereas in these species ADA1 was high. Furthermore, there was no serum ADA2 activity in cattle. Additionally, there were no significant differences between genders in ADA activities in all species. As a result, determination of serum normal ADA and its isoenzyme activities in domestic animals will be useful for scientists who want to investigate ADA activity in future in different diseases.