We cloned a cDNA for a novel granzyme, granzyme N (Gzmn), from a mouse testes cDNA library. The testes contained two distinct species of Gzmn mRNA, one of which codes for a complete protein of 248 amino acids with three essential residues required for catalytic activity. The Gzmn mRNA was specifically expressed in the testes of adult mice. The Gzmn expression was found to initiate in the testes at 3 wk of age and to become more prominent as the animal reached sexual maturity. In situ hybridization analysis revealed that both spermatocytes and spermatids of the adult mouse testes express Gzmn mRNA. Consistent with these findings, the protein was immunohistochemically detected in the spermatocytes and spermatids, although some of the germ cells showed no positive staining. Gzmn was demonstrated to be a secretory and N-glycosylated protein that exists in two protein forms in the testes extract. In the cryptorchid testes, the expression of Gzmn transcript was drastically reduced on Postoperative Day 10, whereas the protein level was gradually decreased starting on Day 6. The local heating (43 degrees C, 20 min) of the testes did not change the Gzmn expression level at either 8 or 16 h after treatment. These results suggest that Gzmn is not involved in the process of germ cell apoptosis induced by heat shock, but that it may be involved in spermatogenesis in the mouse testes.