In order to study the toxic mechanism of action of sulfur mustard (HD), we examined protein expression in control and HD-treated cultured human epidermal keratinocytes (HEK) at 24 h after exposure to 100 μM HD. Protein patterns of cell lysates were prepared using two-dimensional gel electrophoresis with an immobilized pH gradient in the first dimension between 4 and 7 and a molecular weight range in the second dimension between 20 and 200 kDa. Most of the new spots that were observed in the protein profile of HD-exposed HEK, were identified as N- and C-terminal fragments of keratins 14, 16 and 17. These fragments are due to cleavage by caspases, which are proteases responsible for apoptosis, since inhibitors of caspase-3, caspase-6 or of all caspases can prevent the HD-induced keratin degradation. These results indicate that apoptosis is involved in HD-induced cytotoxicity of cultured HEK and that prevention of apoptosis is an option for therapeutic intervention.