HE2, a gene expressed specifically in human epididymis, gives rise to multiple mRNAs that encode a group of small cationic secretory peptides. Localization of HE2 within the defensin gene cluster and prediction that β-defensin-like modules exist suggest that these peptides have antimicrobial activity and represent components of the innate epithelial defense system of the epididymal duct. Reverse transcription-polymerase chain reaction analysis confirmed the occurrence of eight human HE2-derived transcripts, including minor mRNA variants, that had previously been shown only in animal species. Employing isoform-specific antibodies against the predicted HE2 products, multiple 4- to 8-kDa peptides were detected in human epididymal epithelium, epididymal fluid, and ejaculate. N-terminal microsequencing has suggested a proteolytic processing of these peptides by a furin-like proprotein convertase, which cleaves a propiece from the longer precursor peptides. HE2α and HE2β1, representing major peptide isoforms in the human epididymis, were recombinantly expressed, and their susceptibility to furin cleavage was demonstrated in vitro and in vivo. Processed recombinant peptides and chemosynthetic fragments were included in antimicrobial tests. In addition to the β-defensin-like HE2β1 with its expected antibacterial function, HE2α C-terminal fragments showed antibacterial activity against Escherichia coli, although it showed no significant similarity to β-defensins nor to any other known protein family.