Fluorescence microscopy and transmission electron microscopy (TEM) were used to determine the location of the membrane protein PH-20 on spermatozoa of cynomolgus macaques. Rabbit antiserum raised against recombinant cynomolgus macaque sperm PH-20 was used as the primary antibody, and the second antibody was goat anti-rabbit IgG conjugated with either fluorescein isothiocyanate or 15 nm gold particles. Spermatozoa were evaluated before capacitation and after capacitation and induction of acrosome reactions with calcium ionophore A23187. In sperm suspensions with a high percentage of intact acrosomes, fluorescence labeling was observed uniformly over most of the sperm head. The sperm midpiece and tail were not labeled. In sperm suspensions with a high percentage of acrosome reactions, most spermatozoa labeled intensely over the anterior sperm head, but labeling of the posterior sperm head was greatly reduced. TEM of acrosome-intact spermatozoa revealed gold particles distributed uniformly on the plasma membrane overlying the acrosome, the equatorial segment, and most of the post-acrosomal region. After the acrosome reaction, gold label was present on the inner acrosomal membrane and on the plasma membrane overlying the equatorial segment. Very little label was present on the plasma membrane in the post-acrosomal region of acrosome-reacted spermatozoa. The location of PH-20 on the surface of macaque spermatozoa suggests a function for this protein in primary and/or secondary binding to the zona pellucida. The apparent decrease in amount of PH-20 on the posterior head of macaque spermatozoa following the acrosome reaction is consistent with the migration of this protein to the inner acrosomal membrane, as demonstrated previously for the homologous PH-20 protein of guinea pig spermatozoa.