The molecular identity of mouse sperm acrosome antigen recognized by HS-63 monoclonal antibody was analyzed by various biochemical, immunological and molecular biological methods. When its cognate antigen, MSA-63 was isolated from mouse testis by immunoaffinity chromatography, a group of protein spots with wide range of molecular sizes and isoelectric points were identified. Through previous studies, it was established that most of these protein spots were actin-like molecules co-purified with MSA-63 protein from mouse testis. To analyze the molecular size heterogeneity of the isolated MSA-63 proteins, rabbit antisera against a computer-predicted antigenic synthetic peptide (amino acid residue No. 160-171) and a recombinant glutathione S-transferase (GST) fusion protein (GST-63) were raised. These two antisera and those raised against the isolated MSA-63 protein were used as the probes in comparative Western blot assay, indirect immunofluorescent assay and enzyme-linked immunosorbent assay (ELISA). Using ELISA, antisera against GST-63 and computer-predicted antigenic synthetic peptides were shown to cross-react with affinity-isolated MSA-63 protein coated on microwells. However, little immunological cross-reactivity was observed between GST-63 fusion protein and the synthetic peptide. By using a Western blot assay, two major protein bands of 22 and 32 kDa, respectively were commonly detected on mouse testis homogenate strips by both anti-MSA-63 and anti-GST-63. In addition, anti-MSA-63 also recognized several protein bands with molecular masses greater than 35 kDa. The results of this study suggested that the molecular heterogeneity of MSA-63 protein isolated from mouse testis and sperm, is due to a series of post-translational modifications on a single gene product. These modifications may include glycosylations, proteolytic digestions and tight non-covalent associations with other testicular cytoskeletal proteins, such as actins.