A cDNA expressed specifically in late stages of mouse spermatogenic cells during spermatogenesis was cloned by using overlapping RT-PCR and RACE. The cDNA contained an open reading frame (ORF) of 2625 base pairs that encoded an 874 amino acids protein. Comparison analysis of amino acid sequence showed 91% and 80% identity to a rat homologue XP-226242 and a monkey homologue BAB63115 respectively. The expression of the mRNA was only observed in pachytene spermatocytes, round, and elongating spermatids. We named this gene as SRG-L (spermatogenesis related gene expressed in the late stages of spermatogenic cells, GenBank accession No. AY352586). The tissue-specific analysis showed that the SRG-L was highly expressed in spleen and testis. The results suggested that SRG-L might play special roles during spermatogenesis, particularly related to meiosis and spermiogenesis. Analysis of the amino acid sequence showed there was a coiled-coil region near the N-terminal region and rich phosphorylation sites, suggesting SRG-L might function as a transmembrane protein mediating signal transduction. This study also demonstrated that gene cloning by RT-PCR was applicable and convenient when its homologous gene was known.