We used differential display in combination with cDNA cloning to isolate a novel rat gene, designated as Spetex2, that has an open reading frame of 582 nucleotides, encoding a protein of 194 amino acids. Spetex2 mRNA was highly expressed in testis and spleen, and its expression in rat testis was developmentally up-regulated. In situ hybridization revealed that Spetex2 mRNA was predominantly expressed in haploid spermatids at steps 1–13 within the seminiferous epithelium. A BLAST search against rat genome databases at the National Center for Biotechnology Information revealed that the Spetex2 gene is composed of four exons and is mapped to at least 18 loci in a cluster on rat chromosome 15p16, indicating that the genes occur as a repeated tandem array over a long stretch of genomic DNA. By immunocytochemical analysis with confocal laser-scanning microscopy, SPETEX2 protein was detected as a dot-like distribution on the cell periphery of haploid spermatids (steps 1–13) but was not observed in other spermatogenic cells. On the basis of these data, we hypothesize that SPETEX2 might be correlated with cell differentiation of spermaytids in rat testis.