Sulfur mustard [bis(2-chloroethyl)sulfide; SM] is a chemical warfare agent that produces edema and blister formation with a severe inflammatory reaction. The neuropeptide Substance P (SP) contributes to skin inflammation. The mouse ear vesicant model for cutaneous SM injury has been used to evaluate pharmacological agents for countering SM injury. The vanilloid, olvanil has been shown to reduce SM-induced edema and mRNA expression of cytokines and chemokines, suggesting that blocking the inflammatory effects of SP may provide protection against SM-induced dermal injury. This study examined SP expression in skin exposed to SM. Mice were exposed topically to SM (0.16 mg) on the inner surface of the right ear, with or without olvanil pretreatment, and tissues were collected at 1, 10, 30, 60, and 360 min following SM exposure. SP mRNA was localized in skin using in situ hybridization. In naïve skin, mRNA localization was associated with blood vessels and sebaceous glands. In SM-exposed samples, hybridization was also detected in perivascular dermal cells. SP protein expression was identified immunohistochemically in the ear skin of naïve, SM-, olvanil/SM-, and vehicle-treated mice. For the inner surface of the ear, the mean number of SP-positive perivascular cells in the dermis of olvanil-treated/SM-exposed ears was significantly lower than that of SM-exposed ears at both 60 and 360 min. In the outer surface of the ear, the mean number of SP-positive perivascular cells in the dermis of olvanil-treated/SM-exposed ears was significantly lower than that of SM-exposed ears at 360 min. These finding suggest that drugs targeting pro-inflammatory neuropeptides may reduce the severity of SM-induced cutaneous damage.