Using Expressed Sequence Tags (ESTs) deposited in the data banks, a cDNA has been assembled that encodes a protein related to the hyaluronidases from bee venom and mammalian sperm. Expression of this cDNA yielded a polypeptide termed HYAL2, which is located in lysosomes. The HYAL2 protein was shown to have hyaluronidase activity below pH 4. However, it only hydrolyzed hyaluronan of high molecular mass from umbilical cord, rooster comb, and a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which was further hydrolyzed to small oligosaccharides by the sperm hyaluronidase. Conversely, hyaluronan fragments from vitreous humor, which had a molecular mass of about 20 kDa, were not cleaved by the HYAL2 enzyme to any detectable extent. These results provide evidence for the existence of structural domains in hyaluronan, which are resistant to the action of this enzyme. The structural and functional implications of these findings are discussed.