Purpose. Hyaluronic acid (HA) is the predominant glycosami-noglycan (GAG) of the human vitreous. Interaction of this HA with vitreous collagen is important for maintaining gel structure. The mechanism of HA homeostasis in the vitreous is incompletely understood. The aim of this study was to determine whether hyaluronidase, an endoglycosidase that degrades HA, was present in human vitreous.Methods. Vitreous samples were collected from post-mortem eye bank specimens and from non-hemorrhagic, non-inflamed biopsy specimens. Vitreous hyaluronidase was purified by a series of column chromatographic steps, and its activity was measured by an ELISA-like assay and by substrate gel electro-phoresis through an HA-impregnated gel. The purified hyaluronidase was also analyzed by SDS-polyacrylamide gel elec-trophoresis (SDS-PAGE) and by Western blotting.Results. Hyaluronidase activity was detected in vitreous samples from both post-mortem and biopsy specimens. The enzyme was most active at acid pH, but demonstrated significant activity at neutral pH. The partially purified enzyme migrated as a 59 kDa protein on SDS-PAGE, and a single band on Western blots.Conclusions. Hyaluronidase is present in the human vitreous. Thus, hyaluronidase may be involved in HA catabolism in the vitreous and may play a role in determining its gel structure.