Previous studies have shown the implication of β-defensins in host defense of the human body. The human β-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human β-defensin, called human β-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-α (TNF-α), hBD-3 expression is increased particularly after stimulation by interferon-γ. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human β-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.