The sperm plasma membrane protein PH-20 has a hyaluronidase activity that enables acrosome-intact sperm to pass through the cumulus cell layer of the egg. In this study we analyzed the relationship of guinea pig PH-20 and the "classical" soluble hyaluronidase released at the time of the acrosome reaction of guinea pig sperm. PH-20 is a membrane protein, anchored in the plasma and inner acrosomal membranes by a glycosyl phosphatidyl inositol anchor. Several types of experiments indicate a structural relationship of PH-20 and the soluble hyaluronidase released during the acrosome reaction. First, an antiserum raised against purified PH-20 is positive in an immunoblot of the soluble protein fraction released during the acrosome reaction. In the released, soluble protein fraction, the anti-PH-20 antiserum recognizes a protein of approximately 64 kDa, i.e., identical in molecular mass to PH-20 (approximately 64 kDa). Second, the enzymatic activity of the released hyaluronidase is completely inhibited (100%) by the anti-PH-20 antiserum. Third, almost all (97%) of the soluble hyaluronidase is removed from the released protein fraction by a single pass through an affinity column made with an anti-PH-20 monoclonal antibody. These findings suggest that the released, soluble hyaluronidase is a soluble form of PH-20 (sPH-20). During the acrosome reaction, PH-20 undergoes endoproteolytic cleavage into two disulfide-linked fragments whereas the released sPH-20 is not cleaved, suggesting the possible activity of a membrane-bound endoprotease on PH-20. We searched for a cDNA encoding sPH-20 but none was found. This result suggests that sPH-20 may arise from the enzymatic release of PH-20 from its membrane anchor, possibly at the time of acrosome reaction.