A new method that enables the ABO blood type of semen to be determined by identifying the molecular weight of a blood group substance (BGS) has been developed. In order to produce an antibody against a BGS (p 84) detected on the sperm plasma membrand (SPM), p 84 was purified from seminal plasma, injected into a rabbit. When seminal plasma was immunoprecipitated with the anti-p 84 antibody, SDS-PAGE analysis of the immune complex yielded three specific polypeptides with molecular masses of 84, 51 and 25 kDa. The first of these polypeptides, p 84, showed ABH antigenic activity when subjected to immunoblotting. The 51 and 25 kDa proteins corresponded to rabbit IgG heavy and light chains, respectively. Using this method, ABH antigenic activity of a band with a molecular mass of 84 kDa was detected in 25 seminal plasma samples by immunoblotting. When serial two-fold dilutions of seminal plasma were analyzed using this method, clear immunoreactive 84 kDa bands were observed up to 60-fold dilution. When seminal plasma was mixed with vaginal fluid at varying ratios, ABH antigenic activity of 84 kDa immunoreactive bands could be detected until the seminal plasma: vaginal fluid mixture reached a ratio of 1:20. These results suggest that this method will be applicable to the analysis of forensic samples of semen contaminated by vaginal fluid, such as those obtained from victims of sexual assault.