Tesmin is a testis-specific protein. Four mouse tesmin cDNAs so far reported encode a testis-specific, metallothionein-like, 30-kDa protein (tesmin-30). An antibody against tesmin-30, however, detected a protein of 60 kDa (tesmin-60) from the mouse testis. To resolve the relationship between the two, the immunoprecipitated native tesmin-60 was sequenced. The result indicated that tesmin-30 is not full-length but is part of the C-terminal half of tesmin-60. The full-length cDNA (2.2 kilobases [kb]) encoding tesmin-60 (475 amino acid residues) and its genomic DNA (23 kb) were cloned and sequenced. A search of databases indicated that tesmin is a member of the CXC-hinge-CXC family. Immunohistochemistry indicated that tesmin exhibits dynamic subcellular localization changes during spermatogenesis. Before meiosis, it was localized in the cytoplasm of early to late spermatocytes and then translocated into the nucleus just before meiotic division. After meiosis, it appeared in spermatids, starting from the acrosomal vesicles, moving to the nuclear membrane and then to the caudal end as the spermatids elongated, and finally relocating into the cytoplasm. Oxidative stress by cobalt chloride, as well as by diethylmaleate, induced both premature translocation of tesmin from the cytoplasm to the nucleus and apoptotic signals in spermatocytes. The persistent existence of tesmin and its temporally and spatially dynamic localization suggest that tesmin is involved in multiple stages of spermatogenesis and spermiogenesis, possibly during sperm maturation and/or morphogenesis.