By use of differential display in combination with cDNA cloning approach, we isolated a novel rat gene designated as Spergen-3, which has an open reading frame of 2055-length nucleotides encoding a protein of 685 amino acids. Spergen-3 gene is composed of 15 exons and mapped on chromosome 5q36, and its mouse counterpart, which shares 85.5% identity to rat Spergen-3 at the amino acid level, is mapped on chromosome 4E1. Spergen-3 encodes a 75-kDa soluble protein bearing putative 2 EF-hand motifs, proline-repeat, and a putative nuclear localization signal. Of the 2 EF-hand motifs in Spergen-3, the second one seems to match the consensus sequence. Reverse transcription-polymerase chain reaction analysis showed that the expression of Spergen-3 is developmentally up-regulated and that it is exclusively expressed in testis. In situ hybridization revealed that Spergen-3 mRNA was exclusively expressed in haploid spermatids, but its signal was weak or undetectable in spermatogonia, spermatocytes, and Sertoli cells as well as in interstitial cells. We interpreted these data as a potential role of Spergen-3, a new member of EF-hand family, in differentiation of haploid spermatids in testis.