A major regulatory site for species specificity of fertilization in mammals lies at the level of sperm binding to the zona pellucida. This implies a high degree of complementarity between gamete and receptor molecules. These molecules support species-specific interactions between sperm and oocyte that lead to gamete fusion (fertilization). We identified a rat sperm head surface antigen using an IgG1 monoclonal antibody (HD1) against rat sperm of epididymis cauda. By electron microscopy the antigen was shown to be present on the plasma membrane surface of the sperm dorsal head. One- and two-dimensional immunoblotting analysis of sperm proteins demonstrated that HD1 reacted only with a 52 K molecule with a pI ranging from 6.6 to 7.2. The 52 K protein was first detected in situ by indirect immunofluorescence and showed to be underlining few elongated spermatids in testis. However, when the sperm reached the epididymis caput, the antigen was seen to be expressed on the dorsal surface of spermatozoa head. A similar fluorescence reaction was detected on sperms in the epididymis corpus and cauda. The specific spermatozoa-zona pellucida interaction was inhibited in the presence of monoclonal antibody HD1 in a sperm binding assay of in vitro fertilization.