Severe burn causes immunosuppression, and the eschar remains a perfect culture medium for microbial growth. The resulting sepsis is a common complication of burns with a high mortality. The skin produces a number of molecules including antimicrobial peptides (AMPs) that act in the first line of host defense. Previous studies from our laboratory suggested decreased expression of human beta-defensin-2 (HBD-2) in burned wounds. Here, we have expanded our work by identifying HBD-1, HBD-2, HBD-3 and human neutrophil peptide (HNP) in normal and burn skin samples using fluorescence deconvolution microscopy. In normal skin, HBD-1 was localized to the perinuclear region of keratinocytes, while HBD-2 was seen primarily in the stratum basale of the epidermis. HBD-3 was found in dendritic cells of the stratum spinosum. HNP was distributed in a somewhat random pattern in the papillary dermis. In burned skin, in which the epidermis had been destroyed or disrupted, the presence of HBD-1 was localized to dermal glandular structures and hair shafts. HBD-2 was found in both the upper portions of the remaining keratin layers, and localized to lower, f-actin containing, acini-like structures, a pattern also evident with HBD-3. We conclude that although the upper layers of skin are destroyed and disrupted by burn, cells in the lower portions of the skin demonstrate an ability to synthesize most of the AMPs, thereby maintaining some barrier against infection. The results of these studies further contribute to an understanding of the role of AMPs in the pathophysiology of cutaneous burn and the possibility of using these sites for upregulation of AMP synthesis in the prevention of burn sepsis.