A hyaluronidase (EC 3.2.1. 35) was isolated and purified from Agkistrodon acutus venom. The purification procedure included CM-Sephadex C-50 chromatography, gel-filtration on Sephadex G-75 and CM-Sephadex C-25 chromatography. The purified preparation of the enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 4.3, a 45-fold purification being obtained. The hyaluronidase was a glycoprotein (positive PAS staining) with a molecular weight of 33,000 and a pI of 10.3. No hemorrhagic activity was found. The hyaluronidase had an optimum pH of 3.5-5.0 and an optimum temperature of 37 degrees C. It was heat sensitive, was more stable in the acidic than in the neutral region, and lost its activity in the alkaline region. Fe2+, Cu2+ and heparin inhibited the venom hyaluronidase. The Km value for hyaluronic acid was 6.2 X 10(-3) mg/ml.