The inhibiting effect on fertilization of antibodies to the intra-acrosomal protein SP-10 was studied in a bovine in vitro fertilization model. Indirect immunofluorescence showed that antibodies to human SP-10 localized to the acrosomal region of methanol-fixed, but not live, bovine spermatozoa, confirming the intra-acrosomal localization of bovine SP-10. The presence of the SP-10 protein in bovine spermatozoa was further established by immunoblotting of extracts of bovine spermatozoa with monoclonal antibodies and polyclonal antibodies to human SP-10. The antibodies identified a pattern of immunoreactive peptides similar to human, baboon, monkey and pig SP-10. Fertilization experiments in vitro demonstrated that monoclonal antibodies, as well as polyclonal antibodies, to human SP-10 significantly reduced fertilization rates of bovine oocytes by bovine spermatozoa. SP-10 antibodies exerted their anti-fertilization effect by reducing sperm-zona secondary binding. Consistent with the accessibility of the SP-10 antigen following capacitation, SP-10 antibodies also reduced the ability of capacitated spermatozoa to complete the acrosome reaction. Furthermore, SP-10 antibodies affected the motility of capacitated spermatozoa, while not affecting the motility of noncapacitated spermatozoa.