Defensins are important elements in innate immunity that can also trigger adaptive immune responses. The defensins form a family of small cationic antimicrobial peptides with six characteristic cysteine residues, whose pairing pattern in forming three intramolecular disulfide bonds defines the alpha- and beta-defensin subfamilies. In a search for new beta-defensin genes, we performed computational analysis using the Celera mouse genome data base and found exons encoding 23 different beta-defensins, including the eight previously characterized members of this family. Among the new beta-defensins, nine of them form two groups of phylogenetically related sequences that were characterized in greater detail. Northern blot, reverse transcription PCR, and in situ hybridization analysis showed that expression of these genes is restricted to the epididymis, with a specific regional expression pattern. One of the new beta-defensins (Defb38) was chemically synthesized; in in vitro assays on Gram-positive and -negative bacterial strains, Defb38 showed the characteristic salt-dependent antimicrobial activity of beta-defensins. The results demonstrate the existence of a relatively large number of beta-defensins with specific expression in distinct regions of the murine epididymis and suggest complex roles for these proteins in host defense and other physiological processes of the male reproductive tract.