Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K(m) and V(max) values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 microg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 degrees C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe(2+), Mn(2+), Cu(2+) and heparin.