Epithelial β-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human β-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel β-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1β induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is ∼43% identical to HBD-2 and shares the β-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1β. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2β1) encodes a β-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2β1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel β-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.