An acrosomal protein, sp32, was completely purified from acid extracts of ejaculated porcine sperm. Purified sp32 gave a single 32-kDa protein band on SDS-polyacrylamide gel electrophoresis and was characterized as a binding protein specific for 55-, 53-, and 49-kDa forms of (pro)acrosin. This protein was not capable of binding a 43-kDa acrosin intermediate and 35-kDa mature acrosin. sp32 significantly accelerated autoactivation of proacrosin at a basic pH in vitro and affected the maturation pathway of proacrosin. In the presence of sp32, the 49-kDa acrosin intermediate from the 55- and 53-kDa proacrosins was accumulated, instead of the 43-kDa acrosin intermediate. These results suggest that sp32 interacts with both the amino- and carboxyl-terminal sequences of the 53-kDa proacrosin. The cDNA clones coding for porcine and guinea pig sp32 have been identified from testis cDNA libraries in lambda gt11. The deduced amino acid sequence indicates that sp32 is initially synthesized as a 61-kDa precursor protein with a putative signal peptide at the amino terminus. The carboxyl-terminal half of the precursor molecule corresponds to the mature sp32. Thus, sp32 is produced by post-translational modification of the precursor. The binding of sp32 to proacrosin may be involved in packaging the acrosin zymogen into the acrosomal matrix.