Background: Exposure of skin to sulfur mustard (HD) causes rapid separation of epidermal cells from the basal lamina that is visualized as blistering, presumable because proteins that attach epidermal cells to the basal lamina are cleaved by proteases released from injured skin cells and vascular cells. Inhibition of these proteases should theoretically prevent the separation of the epidermal layer and the formation of a blister. Methods: To test this hypothesis, two inhibitors of matrix metalloproteinases (MMPs), ilomastat and doxycycline, and the serine proteases inhibitor, alpha-1 protease inhibitor (A1-PI) were added to explant cultures of human skin that were exposed to sulfur mustard vapor, and 24 hours later the skin explants were assessed histologically for microvesication at the junction of the epidermal layer and basal lamina and for pyknotic nuclei of epidermal cells. Results: No micro-blisters or pyknotic nuclei were detected in skin explants that were not exposed to HD, while large numbers of epidermal cells had pyknotic nuclei and micro-blisters were present along almost the entire epidermis and basal lamina junction in skin explants exposed to HD. Addition of doxycycline (46 micrograms/ml) or A1-PI (2.5 milligram/ml) had no beneficial effect on microvesication. In contrast, no epidermal-dermal separation was observed when ilomastat (80 micrograms/ml) was added to the culture medium. None of the test agents had a positive effect on the extensive HD-induced necrosis in the epidermal keratinocytes. Conclusions: Ilomastat prevents microvesication in this organ cultured human skin model of HD injury. Further testing is needed to assess its effectiveness in reducing blister formation in animal models of skin blistering.