The sperm acrosome contains a variety of hydrolytic enzymes that exhibit differential release during the acrosome reaction. The interaction between hydrolases and the acrosomal matrix, and their compartmentalization into chemically unique matrix domains may represent mechanisms by which this ordered release is achieved. This study characterizes an acrosomal matrix protein that is restricted to the ventral-most region of the apical segment of guinea pig cauda epididymal spermatozoa. Polyclonal antiserum, prepared against an SDS-PAGE-purified apical segment protein, recognized a 50-kDa band, termed AM50, on Western blots. Native AM50 is resistant to solubilization. However, during the acrosome reaction, AM50 is converted into a 42-43-kDa doublet protein (AM50AR) and released into the incubation medium. AM50 and AM50AR are not recognized by antiserum to guinea pig proacrosin and do not exhibit protease activity in a gelatinolytic SDS-PAGE assay. However, AM50AR does bind to p-aminobenzamidine affinity columns, suggesting that it may remain associated with proteases after the acrosome reaction. Light and electron microscopic immunocytochemistry established that AM50 was exclusively localized to the ventral aspect of the apical segment of the acrosome.