Intact human epidermis resists invasion by pathogenic microbes but the biochemical basis of its resistance is not well understood. Recently, an antimicrobial peptide, human β-defensin-2, was discovered in inflamed epidermis. We used a recombinant baculovirus/insect cell system to produce human β-defensin-2 and confirmed that at micromolar concentrations it has a broad spectrum of antimicrobial activity, with the striking exception of Staphylococcus aureus. Immunostaining with a polyclonal antibody to human β-defensin-2 showed that the expression of human β-defensin-2 peptide by human keratinocytes required differentiation of the cells (either by increased calcium concentration or by growth and maturation in epidermal organotypic culture) as well as a cytokine or bacterial stimulus. Interleukin-1α, interleukin-1β, or live Pseudomonas aeruginosa proved to be the most effective stimuli whereas other bacteria and cytokines had little or no ability to induce human β-defensin-2 synthesis. In interleukin-1α-stimulated epidermal cultures, human β-defensin-2 first appeared in the cytoplasm in differentiated suprabasal layers of skin, next in a more peripheral web-like distribution in the upper layers of the epidermis, and then over a few days migrated to the stratum corneum. By semiquantitative Western blot analysis of epidermal lysates, the average concentration of human β-defensin-2 in stimulated organotypic epidermal culture reached 15–70 µg per gram of tissue, i.e., 3.5–16 µM, well within the range required for antimicrobial activity. Because of the restricted pattern of human β-defensin-2 distribution in the epidermis, its local concentration must be much higher. Defensins and other antimicrobial peptides of inflamed epidermis are likely to play an important antimicrobial role in host defense against cutaneous pathogens.