In an effort to examine the molecular basis of spermatogenesis, we isolated two types of novel cDNA clones specifically expressed in mouse testes. Type A cDNA (2,071 nucleotides) is predicted to encode 347 amino acid residues, whereas type B cDNA (1,536 nucleotides) has a deletion of 535 bp from nucleotides 1,206 to 1,740 of type A cDNA, probably due to alternative splicing. This deletion causes a frame shift of the putative open reading frame at the C-terminal portion of type A cDNA to encode 366 amino acid residues. Northern blot analysis using adult ICR organs demonstrated that both types of mRNAs were specifically expressed in testis, although type B mRNA was more abundant than type A mRNA. RT-PCR analysis revealed that these two mRNAs were also expressed in immature testes at 1, 5, 11, 16 and 24 days after birth. In situ hybridization analysis of adult ICR testes demonstrated that these two mRNAs were expressed in spermatogonia, Sertoli cells and Leydig cells. In the W/ WV mouse testis which lacks c-kit activity and spermatogonia, but contains Sertoli and Leydig cells, both mRNAs were found to be expressed in the latter two types of cells. We therefore termed these novel clones tsec-1, testis-specifically expressed cDNAs-1. The protein products of tsec-1 may play an important role in mammalian spermatogenesis.