We identified a testis-specific protease-like protein tentatively named TESPL and a pancreatic trypsinogen Prss2 from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. The enzymatic motifs and the cysteine patterns in serine proteases are highly conserved in these two proteins. Based on the phylogenetic analysis, Prss2 duplicated recently and TESPL underwent distant evolution without gene duplication from the progenitor of trypsin-like and chymotrypsin-like proteases. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. In contrast, Prss2 was constitutively expressed in many tissues including testis. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region. Further, orthology group for mouse TESPL was identified in the conserved gene family of eutherian testis serine protease 5. J. Cell. Biochem. 110: 620–629, 2010. © 2010 Wiley-Liss, Inc.