Our laboratory studies the mechanism of toxicity of the chemical blistering agent sulfur mustard (SM) for the purpose of developing medical countermeasures to this chemical threat agent. SM is a cytotoxic alkylating agent with mutagenic and vesicating properties. The exact mechanism of SM-induced skin vesication is not completely understood. The human epidermal keratinocyte (HEK) in culture is the in vitro equivalent of the basal epidermal cell of human skin, a principal target for the toxicity of SM in tissue. In this report, our laboratory evaluated several markers of cytotoxicity in HEK exposed to SM and a variety of chemical toxicants. The markers studied were protein content, cellular morphology, membrane integrity, annexin V labeling and DNA fragmentation. The chemical toxicants were calcimycin (A23187), staurosporine (STR), cycloheximide (CHX), actinomycin D (ActD) and camptothecin (CPT). All six compounds lowered cellular protein levels at 24 hours post-exposure, with A23187 and STR having significant effects as early as 4 hours after exposure. A23178 caused an early (1 hr) annexin V response, but these cells rapidly die as determined by an uptake of propidium iodide (PI). By 4 hours, only STR shows an increase of annexin positive cells. SM failed to generate annexin staining at either 4 or 24 hours post-exposure. As for morphological changes, only STR and A23187 led to changes at 4 hours. STR resulted in stellate-shaped cells, whereas A23187 yielded cells that rounded up and had contracted cytoplasm. At 24 hours, A23187 caused DNA containing cytoplasmic blebs, whereas SM-exposed cells had blebbing without DNA content. Comet analysis of DNA fragmentation did not discriminate different types of death processes by the different toxicants. These results help elucidate the contributions of various death processes following toxicant exposure and hopefully will contribute to a better understanding of critical events in SM pathology.