To identify new genes that are turned on following the expression of a pre-T-cell antigen receptor in early thymocytes, we developed a PCR-based subtractive approach that selects for mRNAs that are differentially expressed between thymocytes at the CD4(-)CD8(-) and CD4(+)CD8(+) stages. This approach led to the characterization of a cDNA, Tssn1, which is predominantly expressed in CD4(+)CD8(+) cells and at specific stages of spermatogenesis. We analyzed the structure of the Tssn1 gene in mouse and human and found that it is highly conserved. Of interest, Tssn1 is adjacent to an unrelated gene named 2310047L21Rik and gives rise to hybrid 2310047L21Rik--Tssn1transcripts. Therefore, this analysis offers insights into the critical steps that may precede the birth of new genes endowed with either compound functions or novel tissue distribution.