Development of spermatozoa is a complex process involving specific morphological formation of flagella, nucleus and mitochondria. Although detailed morphological observations of these events are available, the molecular mechanisms remain to be fully elucidated. We report here the molecular cloning and characterization of mouse spergen-1 encoding a sperm specific mitochondrial protein, from a haploid germ cell-specific subtracted cDNA library of mouse testis. Isolated cDNA is c. 0.7 kb and contains a 465 bp ORF that encodes mouse spergen-1, a sperm mitochondrial protein consisting of 154 predicted amino acids. Antibodies raised against mouse Spergen-1 identified a testis-specific c. 18 Mr x 10(3) band in Western blot analysis. The protein was localized to the mitochondria of mouse sperm. Comparison of the mouse and human genomic sequences showed that 55 bps of the 5'-upstream region containing a CAAT box and binding sequence for NF-kappa B is conserved and could be important for specific expression of mouse spergen-1 in haploid germ cells.