To identify a novel candidate(s) for acrosomal proteins that act on the sperm/egg interaction, a DNA fragment was PCR-amplified from a cDNA library of acrosin-deficient mouse testis and then used as a probe to screen a mouse testis cDNA library. Complementary DNA clones encoding each of two similar but different serine proteases, TESP1 and TESP2, have been identified. The nucleotide sequences of these clones indicate that mouse TESP1 and TESP2 are initially synthesized as preproproteins of 367 and 366 amino acids, respectively. Comparison of the two TESP sequences with those of typical serine proteases suggests that each TESP zymogen is probably converted into a two-chain mature enzyme consisting of light and heavy chains covalently linked by a single pre-existing disulfide bond. The conversion may be accomplished by another protease(s) with a trypsin-like cleavage specificity, since it is unlikely that the mature TESP1 and TESP2 are capable of splitting the Lys-Ile bond between the light and heavy chains. Northern blot analysis of total cellular RNA demonstrates that the TESP1 and TESP2 genes are expressed only in the testis, and the transcripts are abundantly present in the haploid round spermatids. Moreover, immunocytochemical analysis of mouse cauda epididymal sperm using affinity-purified antibodies reveals that these two TESPs are both localized in the sperm acrosome and are released during the acrosome reaction induced by calcium ionophore A23187. These findings provide additional clues for elucidating the mechanisms involved in the sperm/egg interactions, including penetration of the zona pellucida by sperm.