In vertebrate tissues, cell integrity is maintained by at least three mechanisms. During an immune response, injured cells are eliminated by cytotoxic lymphoid cells that produce perforin, granzyme B, and Fas ligand (FasL). Second, epithelial cells can produce FasL as an immunosuppressive protein, probably to protect the tissue against immune-mediated damage. Third, locally secreted antimicrobial peptides can be operative in the protection of animal and human epithelia. In this work, as another contribution to local mechanisms of host defense, the ability of human epidermal keratinocytes to produce cytotoxic proteins was investigated. To address this question, freshly isolated human epidermal cells and keratinocytes grown in vitro were studied. Freshly isolated epidermal cells did not express the cytolytic proteins. In contrast, keratinocyte growth to confluence was associated with granzyme B, perforin, and FasL mRNA and protein synthesis. These proteins were secreted in the culture medium. Further analysis showed that they were identical with the ones used by cytotoxic lymphocytes. Their function was then investigated with a view to a potential role in epidermal cell integrity. The data showed that activated human keratinocytes were able to protect against invading pathogens through granzyme B expression. This was demonstrated by the ability of granzyme B to greatly decrease the bacterial growth of Staphylococcus epidermidis. In addition, keratinocytes expressing FasL were found to prevent immune epidermal cell damage. Apoptosis of Fas-sensitive T cells occurred during coculture with confluent epidermal keratinocytes and was largely reduced by the addition of a FasL inhibitor. The data favor keratinocyte involvement in the regulation of dermal inflammatory responses.