Monoclonal antibodies (mAbs) against spermatozoa are a popular approach to define sperm antigens involved in the process of fertilisation. The identification and characterisation of a 57-kDa fertility asssociated sperm antigen (FASA-57) from human spermatozoa was reported in an earlier paper by the authors. In the present report, studies to develop mAbs against partially purified FASA-57 are extended. From a panel of mAbs raised, one clone designated as 3H₄B₉ was selected and characterised because it recognised native FASA-57. Indirect immunofluorescence studies revealed that FASA-57 localised on the acrosome of non-acrosome-reacted human spermatozoa and on the equatorial region after the acrosome reaction. Spermatozoa from several other mammalian species were also found to express this antigen, suggesting its evolutionary conservation across the species. The antigen localised specifically in spermatogonial cells and luminal spermatozoa of the testis and epididymis. Western blot studies showed the presence of a FASA-57-like protein in the mouse brain also, indicating that testis and brain share antigenic similarities. Further, the role of FASA-57 in sperm–egg interaction was investigated using a mouse model. The mAb 3H₄B₉ inhibited sperm–egg binding and fusion in a dose-dependent manner with half-maximal inhibition at 2 µg mL^–1. In conclusion, FASA-57 appears to play an important role in sperm–egg recognition, fusion and fertilisation. Therefore, FASA-57 could be used as a diagnostic marker in the evaluation of male infertility.