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First identified in psoriatic epidermis and subsequently in other inflammatory cutaneous lesions, human β-defensin-2 (hβD-2) is one of two endogenous antimicrobial peptides related to defensins in plants and animals. Our objective was to determine the expression of hβD-2 after injury and in chronic wounds. Biopsies of normal ipsilateral thigh skin and wound edges were taken from nine consecutive patients with venous leg ulcers (day 1) and from the same biopsy sites 2 days later (day 3). Sequential samples were also obtained from intact or meshed bilayered bioengineered skin consisting of neonatal human keratinocytes and dermal fibroblasts in a collagen matrix. Specimens were processed and immunostained for hβD-2 using a polyclonal rabbit antibody. In both human tissues and bioengineered skin, staining for hβD-2 was confined to the upper epidermal layers, sparing the basal cells. Analysis of 26 tissue samples from patients showed that normal skin had no hβD-2 expression but that marked up-regulation occurred after wounding by day 3. Conversely, chronic ulcers showed moderate-to-strong immunostaining for hβD-2 at baseline on day 1, with little or no change in intensity after wounding by day 3. In vitro, bioengineered skin showed increased distribution of cytoplasmic hβD-2 immunostaining after meshing. We conclude that the expression of hβD-2 is up-regulated after injury. Chronic wounds uniformly show a constitutively high baseline expression of hβD-2, possibly due to ongoing tissue injury and bacterial colonization.