Hyaluronidase is a component of the acrosome of bovine spermatozoa. Utilizing fluorescein and peroxidase-labelled antibodies, the enzyme has been localised at both the light and electron microscopic levels. Antibodies against purified bovine testicular hyaluronidase were prepared in New Zealand white rabbits. Immunodiffusion, immunoelectrophoresis, and immuno-inhibition studies of the antiserum demonstrated the presence of an antibody specific for bovine sperm hyaluronidase with no cross reactivity to other sperm-associated antigens. Fluorescence microscopy of ejaculated bovine spermatozoa show the enzyme to be limited to the anterior portion of the acrosome with a sharp termination at the rostral border of the equatorial segment of the acrosome. Light microscopic studies with peroxidase-labelled antiserum were similar to the fluorescent findings. Fine structure studies revealed reaction product predominately associated with the matrix of the acrosome cap. Within the acrosome, more intense localisation was observed in the apical densities, while no product was visualised within the equatorial segment. These findings are compatible with the hypothesis that the enzyme functions to facilitate the passage of spermatozoa between cells.