Human beta-defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel beta-defensin genes (DEFB25-DEFB29) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of beta-defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation. We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense.