Acrosin is a serine proteinase located in the acrosome of the sperm in a zymogen form, proacrosin. As deduced from the cDNA sequences of human, boar, and mouse proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence of 15 to 18 amino acid residues. We have isolated the gene coding for mouse proacrosin from a mouse cosmid library, using cDNA clones as probes. The gene comprises six exons, and one of the five introns is located in the 5'-untranslated region. The transcription initiation site of the preproacrosin mRNA could be assigned to the residue T, 581 nucleotides upstream of the translation initiation codon ATG, with primer extension analysis. TATA and CAAT boxes could be identified at positions -26 and -97, respectively. Similar to other serine proteases, the coding sequence encompasses five exons and the three active-site residues His, Asp, and Ser are encoded by three different exons (E2, E3, E5). The proline-rich domain, which is a characteristic feature of the proacrosin polypeptide, is encoded in exon 5 with the serine active-site residue. The gene is located on chromosome 15 of the mouse genome, bands E/F, and is a member of a syntenic group that was mapped on human chromosome 22, q13-qter. During spermatogenesis the proacrosin gene in the mouse is expressed diploid, in contrast to a haploid expression observed in bull, boar, and rat.