Antisperm antibody binding to acrosin was investigated by Western Blotting. The clinical significance of this binding specificity was assessed in a 2-year clinical follow-up. Consecutive serum samples positive for antisperm antibodies by both enzyme-linked immunosorbent assay and immunobead testing were evaluated for acrosin-binding specificity. The patients were followed in an outpatient setting by private infertility specialists. Sixty-five consecutive infertile referral patients with positive antisperm antibody were evaluated. Clinical follow-up was obtained on 8 of 9 females with evidence of antibody binding to acrosin and 19 of 26 females with no specific binding to acrosin. Prednisone therapy was given during six courses of intrauterine insemination with husband's sperm. All treatment decisions were made by private physicians independent of the acrosin-binding result. Pregnancy status was obtained as part of a 2-year follow-up. Acrosin-binding specificity was demonstrated in 10 (15%) of the 65 patients. Two of the 8 women (25%) with antibody binding to acrosin and 6 of the 19 women (32%) with antisperm antibodies but no specific binding to acrosin delivered normal children. Although antibody-binding specificity to acrosin could be demonstrated, a 2-year clinical follow-up showed no difference in pregnancy rates when compared with women with antisperm antibodies showing no binding specificity to acrosin.