In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA. Zymogram analysis of L. intermedia venom showed HA lytic activities at 41 and 43kDa, and, when CS was used as a substrate, zymograph experiments resulted in 41 and 43kDa lytic zones. Thus, these results support the hypothesis that the same molecules are involved in cleaving HA and CS residues. Experiments to compare L. intermedia electrostimulated venom and venom gland extract also demonstrated very similar HA lytic activity, suggesting again that hyaluronidases are self-components of Loxosceles spider venom instead of oral egesta contamination. HA degradation as a function of pH in these hydrolase enzymes showed no apparent activities at low or high pH, with optimal activity at 6.0-8.0 pH. Finally, we confirmed the cleaving action of the venom hyaluronidases on HA in the extracellular matrix of the dermis of rabbit by fluorescence reaction to HA and confocal microscope analysis. Thus, hyaluronidases type hydrolases endo-beta-N-acetyl-d-hexosaminidase are implicated as self-components of Loxosceles spider venom and can be involved in venom effects as spreading factors.