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The possible role of the human corneal endothelium in the turnover of anterior chamber hyaluronic acid (HA) was investigated. Hyaluronidase, an endoglycosidase that degrades HA and other glycosaminoglycans, is thought to play a role in HA homeostasis. The presence of hyaluronidase in the corneal endothelium was demonstrated immunohistochemically in sections from normal adult human cornea. Additionally, by using a modified enzyme-linked immunosorbent assay-like assay, active hyaluronidase was detected in the supernatant from primary culture human corneal endothelial cells. The optimal activity for the corneal endothelial hyaluronidase was in the acid range (pH 4.0), similar to previously isolated lysosomal hyaluronidase. Further immunohistochemistry showed that the corneal endothelial cells also express CD44, the receptor for HA, which would allow endocytosis of HA. Human corneal endothelial hyaluronidase may play a role in normal anterior segment HA metabolism and in the degradation of highly concentrated HA used as a viscoelastic.