Complement-dependent sperm-immobilizing antibodies (SI-Abs) have frequently been detected in the sera of infertile women. Analyses using monoclonal antibodies as well as patients' antisperm antibodies have shown that the carbohydrate moieties of sperm and seminal plasma are major epitopes of these antibodies. In the present study, we characterized the antigen molecules recognized by human monoclonal SI-Ab (MAb H6-3C4) derived from a patient with strong SI-Ab activity. Immunofluorescent staining showed that MAb H6-3C4 reacted with ejaculated human sperm and the epididymis but not with the testes and other somatic organs including the spleen. In Western blot analysis, MAb H6-3C4 reacted to antigen molecules of Mr 15-27 kDa present in the methanol-chloroform extracts of either ejaculated sperm or seminal plasma. Amino acid sequence analysis of the reactive molecule revealed that the amino acid sequence was identical to the core peptide of CD52, a GPI-anchored protein on lymphocytes. Furthermore, the epitope for MAb H6-3C4 was found to be the specific N-linked carbohydrate for the male reproductive tract. These results suggest that SI-Abs in some infertile women target a sperm-specific carbohydrate antigen on CD52 molecules which are secreted from the epididymis and coat the sperm surface.