In previous studies, we identified a new member of the male reproductive tract subgroup within family 2 cystatins, termed cystatin 12 (Cst12, previously known as Cst TE-1 or Cres3). The mouse Cst12 mRNA was primarily localized to the Sertoli cells in the testis and to the epithelial cells of the proximal caput region of the epididymis. In this report, studies were carried out to characterize the cystatin 12 (CST12) protein in mouse testis and epididymis. A recombinant His-CST12 fusion protein was expressed in E. coli and purified to generate an anti-CST12 polyclonal antibody. Western blot analysis showed little or no cross-reaction between the anti-CST12 antibody and several other known male reproductive tract cystatins. Immunohistochemistry revealed that CST12 protein was predominantly localized to the cytoplasm of Sertoli cells in the seminiferous epithelium in a stage-dependent manner. All stages showed high levels of expression except stages VII and VIII, in which very limited expression of CST12 was observed. In the epididymis, CST12 was highly expressed in the cytoplasm of the epithelial cells in the proximal caput and secreted into the lumen. The mouse CST12 protein was also detected in other regions of the epididymis; however, the localization varied greatly along the epididymal tubules. Indirect immunofluorescence showed that CST12 protein was localized to the cytoplasmic droplets in both testicular and epididymal spermatozoa. These observations suggest that CST12 protein may play a specialized role during spermatogenesis and sperm maturation. Northern blot analyses demonstrated that Cst12 transcript levels in the epididymis decreased after castration, and testosterone propionate (T) treatment further repressed the expression of this gene. However, 17-beta estradiol (E) administration maintained the expression of Cst12 mRNA after castration, whereas treatment with both T and E failed to maintain Cst12 mRNA levels in epididymis. These results suggest that androgen and estrogen, probably with other testicular factors, are involved in the regulation of this gene.