To elucidate the molecular mechanisms involved with spermiogenesis in testis, we performed differential display screening to isolate genes that are developmentally up-regulated during rat testis development. One of the cDNAs isolated by differential display was highly expressed in testis. Both reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression level of the gene developmentally increased. By screening the rat testis cDNA library, we successfully isolated rat cDNA clones encoding the entire open-reading frame of 462 base pairs coding a small protein of 154 amino acids. Because in situ hybridization revealed that the gene was specifically expressed in haploid spermatids in the rat seminiferous tubules, it was designated as spergen-1 (spermatogenic cell-specific gene-1). The recently opened database of the full-length mouse cDNA collection contains a mouse gene that is homologous to rat spergen-1. Subcellular fractionation followed by immunoblot analysis revealed that spergen-1 protein was associated with mitochondria. The transfection experiments performed in COS-7 cells suggested that spergen-1 has a N-terminal mitochondria-targeting signal. We suggest that spergen-1 might be involved in spermiogenesis by transiently associating with spermatid mitochondria.