Differential display-reverse transcriptase-polymerase chain reaction was used to examine Sertoli cell gene expression. As a result, two new members of the mouse cystatin multigene family were isolated and named cystatin SC (cystatin-related gene expressed in Sertoli cells) and cystatin TE-1 (cystatin-related gene highly expressed in testis and epididymis). The full-length cDNA sequence of cystatin SC contains an open reading frame that encodes a putative signal peptide of 20 amino acids and a mature protein of 110 amino acids, whereas that of cystatin TE-1 encodes a 128 amino acid protein with a predicted signal peptide of 21 amino acids. Both of the deduced amino acid sequences contain four highly conserved cysteine residues in precise alignment with other cystatin family members. The derived cystatin SC and TE-1 amino acid sequences lack some of the specific, highly conserved motifs believed to be necessary for cysteine proteinase inhibition activity. Northern blot analysis revealed that cystatin SC mRNA was detected only in the testis, whereas the cystatin TE-1 gene was highly expressed in testis and epididymis with very low expression in ovary and prostate. In situ hybridization showed that cystatin SC mRNA was localized mainly to Sertoli cells with an obvious stage-dependent expression, and that cystatin TE-1 mRNA was predominantly expressed in Sertoli cells without apparent stage-dependent expression. Cystatin TE-1 mRNA, as displayed by in situ hybridization, was expressed only in the epithelial cells of the proximal caput region of the epididymis. The unusual amino acid sequence and highly restricted expression suggests that cystatins SC and TE-1 play a very specialized role in the testis and epididymis.