Sulfur mustard (SM (bis-(2-chloroethyl) sulfide)), a vesicant, is a chemical warfare as well as a potential terrorism agent. SM-induced skin blistering is believed to be due to epidermal-dermal detachment as a result of epidermal basal cell death via apoptosis and/or necrosis. Regarding the role of apoptosis in SM pathology in animal skin, the results obtained in several laboratories, including ours, suggest the following: (a) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic-necrotic continuum and (b) inhibiting apoptosis decreases SMinduced microvesication in vivo. To study the mechanisms of SM-induced apoptosis and its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multi-well plates (24-, 96-, or 384-well) and high throughput applications. This assay allows replication and multiple types of experimental manipulation in sister cultures so that the apoptotic mechanisms and the effects of test compounds can be compared statistically. SM affects diverse cellular mechanisms, including endoplasmic reticulum (ER) Ca2+ homeostasis, mitochondrial functions, energy metabolism, and death receptors, each of which can independently trigger apoptosis. However, the biochemical pathway in any of these apoptotic mechanisms is characterized by a pathway specific sequence of caspases, among which caspase-3 is a key member. Therefore, we exposed 80-90% confluent HEK cultures to SM and monitored apoptosis by measuring the fluorescence generated due to hydrolysis of a fluorogenic caspase-3 substrate (acetyl- or benzyl oxycarbonyl-Asp-Glu-Val-Asp-fluorochrome, also designated as AC-or Z-DEVD- fluorochrome) added to the assay medium. Fluorescence was measured using a plate reader. We used two types of substrates, one (Sigma-Aldrich, CASP-3-F) required cell disruption and the other (Beckman-Coulter CellProbeTM HT Caspase-3/7 Whole Cell Assay Kit) was cell permeable. The latter substrate was useful in experiments such as determining the time-course of apoptosis immediately following SM exposure without disruption, e.g., due to cell processing. In SMexposed HEK, fluorescence generated from the fluorogenic caspase-3 substrate hydrolysis increased in a time (0 – 24 hr) and concentration (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 mM) dependent manner. SM caused maximum fluorescence at about 0.5 mM. However, at 2 mM SM, fluorescence decreased compared with 0.5 mM, which remains to be explained. Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo, a small fluorescence increase was observed at 6 to 8 hr, which was markedly higher at 12 hr. At 24 hr, all SM concentrations increased fluorescence. Fluorescence increase due to SM was prevented 100% by a caspase-3 specific peptide inhibitor (AC-DEVD-CHO), but less effectively by a general caspase inhibitor (Z-VAD-FMK), indicating that the fluorescence increase was due to caspase-3-mediated apoptosis. These results suggest potential applications of this method to study apoptosis mechanisms and possibly those involving other caspase substrates.