The epithelial surface of the human small intestine is a barrier between the host and the microbial environment of the lumen. A human small intestine cDNA clone was found to encode a new member of the defensin family of antimicrobial peptides, named human defensin-6. Tissue expression of this mRNA is specific for the small intestine as determined by Northern blot analysis and polymerase chain reaction analysis. In situ hybridization demonstrated that human defensin-6 mRNA localizes to Paneth cells in the crypts of Lieberkühn. The finding of an abundant defensin mRNA in human Paneth cells supports the notion that these epithelial cells may play a key role in host defense of the human bowel. The results also strengthen the hypothesis that peptide-based host defenses are prevalent at mucosal surfaces in mammals.