PH-20 is a sperm plasma-membrane protein that has been shown to have hyaluronidase activity in several mammalian species including nonhuman primates. In this investigation, the PH-20 protein was characterized in noncapacitated human sperm and in capacitated human sperm. Two forms of PH-20 were observed in immunoblots of sodium dodecylsulfate polyacrylamide-gel electrophoresis (SDS PAGE) using a polyclonal antibody to recombinant PH-20: a major band of 64 kDa appeared in noncapacitated and capacitated sperm extracts and a 53-kDa band that appeared only in the acrosome-reaction supernatant of acrosome-reacted sperm. Using hyaluronic acid substrate gel analysis, we demonstrated that noncapacitated sperm extracts, capacitated sperm extracts, and the acrosome-reaction supernatant had hyaluronidase activity at neutral pH (pH 7) and acid pH (pH 4). The 64-kDa form in all samples had hyaluronidase activity at both neutral and acid pH, but the 53-kDa form was only active at acid pH. Total hyaluronidase activity, as measured by a microplate assay, was higher at pH 7 than at pH 4. Very low hyaluronidase activity was detected in the acrosome-reaction supernatant. Transmission electron microscopy and immunogold labeling showed that PH-20 of acrosome-intact human sperm was located on the plasma membrane over the entire head but not on the sperm midpiece and tail. After the acrosome reaction, PH-20 was also located on the inner acrosomal membrane. The biochemical characteristics and the ultrastructural localization of PH-20 in human sperm suggest that this protein is the human sperm hyaluronidase and, therefore, has an important function during fertilization.