Cutaneous exposure to sulfur mustard (bis(2-chloroethyl) sulfide, HD), a chemical warfare agent, produces a delayed inflammatory skin response and severe tissue injury. Despite defined roles of inflammatory cytokines produced or released in response to skin-damaging chemicals, in vivo cytokine responses associated with HD-induced skin pathogenesis are not well understood. Additionally, there is little information on the in vivo temporal sequence of gene expression of cytokines postexposure to HD. The goal of these studies was to identify in vivo molecular biomarkers of HD skin injury within 24 hours after HD challenge. Gene expression of interleukin 1b (IL-1b), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 6 (IL-6), and interleukin 1␣ (IL-1␣) in the mouse ear vesicant model was examined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). An increase in IL-1b mRNA levels was first observed at 3 hours. IL-1b, GM-CSF, and IL-6 mRNA levels were dramatically increased at 6-24 hours postexposure. IL-1␣ mRNA levels were not increased following HD exposure. Immunohistochemical studies demonstrated that IL-1b and IL-6 protein was produced at multiple sites within the ear, including epithelial cells, inflammatory cells, hair follicles, sebaceous glands, the dermal microvasculature, smooth muscle, and the dermal connective tissue. An increase in the intensity of staining for IL-1b and IL-6 was observed in localized areas at 6 hours and was evident in multiple areas at 24 hours. Positive staining for GM-CSF immunoreactive protein was localized to the inflammatory cells within the dermis. The number of immunostaining cells was increased as early as 1 hour following HD exposure. These studies document an early increase in the in vivo expression of inflam-