For successful fertilization to occur, mammalian spermatozoa must undergo a series of modifications in order to reach and penetrate the oocyte. Some of these modifications occur during passage through the epididymis, the site where spermatozoa acquire their fertilizing ability. We have previously described hamster sperm protein, P26h, which is acquired by spermatozoa during epididymal transit, and have proposed that this protein is involved in sperm-egg binding. In the present study, we report the cloning and characterization of the full-length cDNA encoding hamster P26h. A database search using the predicted hamster P26h amino acid sequence revealed 85% identity with mouse AP27 protein and porcine carbonyl reductase, members of the short-chain dehydrogenase/reductase (SDR) family of proteins. Northern blot analysis revealed a major P26h 1-kilobase transcript in the testis. No signal was detected in other somatic tissues of the hamster. In situ hybridization experiments revealed that the P26h gene was predominantly transcribed in seminiferous tubules of the testis and at a lower level in the corpus epididymidis. The identity of the cloned P26h was confirmed by immunoprecipitating in vitro-translated P26h using polyclonal antiserum raised against purified hamster sperm P26h. Taken together, these results identify P26h as a new member of the SDR family of proteins involved in the processes of mammalian gamete interactions.