Sulfur mustard (HD) is a chemical warfare agent that has been used during World War I, and more recently in conflicts in the Middle East. This highly toxic blistering agent causes severe dermal, gastrointestinal, respiratory and ocular injuries. HD-induced cutaneous lesions have been fairly well characterized using both in vitro and in vivo model systems. Normal human epidermal keratinocytes (keratinocytes) have been used as an in vitro model to study cutaneous injury by HD, and for screening of potential anti-inflammatory antidotes. In spite of its high sensitivity to HD vapors, little is known regarding ocular tissue injury, which may range from severe corneal damage to even loss of vision. The early molecular and biochemical events leading to HD ocular pathological effects, and the biochemical markers of these pathological effects have not been explored. In this study, we examined the induction of pro-inflammatory mediators in human corneal epithelial cell (HCEC) cultures upon exposure to nitrogen mustard (NM). NM is closely related chemically and toxicologically to HD. Cytotoxicity of NM to HCECs and keratinocytes was compared by cell viability assay using a tetrazolium reagent (MTS). Results indicate that HCECs are more susceptible (LC50=10 μM) to NM when compared to keratinocytes (LC50=50 μM). Cytokine release in HCEC culture supernatant during exposure of up to 25 μM NM was measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Dose dependent secretions of interleukin-6 (IL-6) and IL-8 in the culture supernatants of HCECs were observed during exposure to NM; four-fold increases in the secretion of these two cytokines were detected upon treatment with 25 μM NM for 24h. No changes in the levels of IL-1β, TNF-α, and IL-12 p40 were observed in NM-treated HCECs. Further, gelatin zymography for matrix metalloproteinases revealed the increased secretion of MMP-2 and MMP-9 in NM-exposed HCEC culture supernatant. Changes in the expression levels of MMPs and cytokines were also evaluated by reverse transcriptase-polymerase chain reaction studies. The observed increase in the secretion of cytokines and MMPs at lower doses of NM compared with keratinocytes corroborates with the higher sensitivity of HCECs determined using the cell viability assay. This study suggests that the responses of HCECs to vesicating agents as defined by cytokine and MMP secretion, are comparable to those observed with keratinocytes. Therefore, HCECs are a viable in vitro model to evaluate the molecular and biochemical events leading to HD ocular injury and for screening of potential anti-inflammatory antidotes.