This is an overview of the biochemistry, biological function and therapeutic uses of hyaluronidase and its substrate, hyaluronate. We focus on the role of hyaluronate and its receptor CD44 in cell–cell and cell–matrix adhesion and cell activation as well as on the putative role of hyaluronate and hyaluronidase in morphogenesis. Variants of CD44 and their putative role in tumor metastasis are also included. Other topics that are discussed are the chemical and enzymatic nature of hyaluronidase, i.e. the mode of substrate degradation, pharmacodynamical and pharmacokinetic aspects of this enzyme and its role as spreading factor. Purification methods, possible contaminations and techniques of activity determinations are mentioned as well as the physiological role of hyaluronidase and tumor-associated alterations in serum and tissue enzyme levels. As far as therapeutic applications are concerned, we discuss uses of hyaluronidase in ophthalmology and regional anesthesia as well as pain management in osteoarthritis using hyaluronate.