The enzyme hyaluronidase found in the venom of many spiders degrades hyaluronic acid (HA), a glycosaminoglycan in the extracellular matrix of connective tissues, and thereby acts as a spreading factor for the spider toxins in the prey. So far there are no details available of the structure of this enzyme from the venom of member of the sub-phylum Arachnida. We have identified the presence of this enzyme in the venoms of the Brazilian spiders Ancylometes sp. (ANC), Phoneutria keyserlingi (PK), Phoneutria nigriventer (PN) and Oligoctenus ornatus (OCT) by a zymographic method in which the substrate (HA) was incorporated into 12.5% SDS-PAGE gels. The concentration of the enzyme was found to be highest in the venoms of ANC and female OCT spiders. In all cases the molecular mass of the enzyme was approximately 37 kDa. With the objective of determining the primary structure of this enzyme, we are currently purifying the HA enzyme by subjecting the crude venoms to reverse phase chromatography (RPC) on HPLC columns of Vydac C₄ and using extended linear gradients of acetonitrile in 0.1% aqueous TFA. The active fractions, identified by HA-SDS-PAGE zymograms, were then further purified by anion-exchange FPLC on Resource Q columns using a linear gradient of 0-0.3M NaCl in 10mM Tris-HCl at pH 8.6.