The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.