Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2, 2’-dichlorethylsulfide (sulfur mustard, military designation HD) and 2, chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-α) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100™ , and reverse transcription-polymerase chain reaction (RTPCR). The messenger RNA expression of hTNF-α was determined to provide a semiquantitative analysis. HD-stimulated NHEK induced secretion of hTNF-α in a dosedependent manner. Dose response effect of Lewisite decreased hTNF-α levels. Timeresponse data indicated that the maximum response for HD occurred at 24 hr with an associated cytotoxic concentration of 10-4 M. NHEK cells stimulated with 10-4 M HD for 24 h at 37 οC increased detectable levels of hTNF-α from 5 to 28 ng/mL at an index of cell viability between 85 to 93 % as detected by Luminex100™. Our results indicated that the increased levels of hTNF-α by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-α from control levels of 1.5 ng/mL to 0.3 ng/mL after stimulation (10-4 M), with an index of cell viability of ~ 34 %. We analyzed the intracellular signaling pathway leading to the transcriptional control of hTNF-α gene. We reported that HD in cultured NHEK activates hTNF-α gene and that L at 10-6 to 10-4 M markedly reduced hTNF-α expression. We conclude that the inflammatory mediator, hTNF-α, could be a potential biomarker for differentiating between exposure of HD or L.