Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.