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Sulfur mustard (SM), chemically 2,2′-dichloro diethyl sulphide, is an incapacitating and extremely toxic chemical warfare agent. It causes serious blisters on contact with human skin. While screening various antidotes against its toxicity, we observed that SM was more toxic through percutaneous (p.c.) route compared to oral (p.o.) and subcutaneous (s.c.) routes. The LD50 of SM in female mice was found to be 5.7, 8.1 and 23.0 mg/kg through p.c., p.o., and s.c. routes, respectively. The body weight of the animals was monitored and it was found that percentage body weight loss was more in the p.c. route. There was significant DNA fragmentation in liver in all the three routes evaluated at 19.3 mg/kg dose of SM. The depletion of hepatic GSH content was found to be more in the p.c. route of exposure compared to s.c. route. There was significant reduction in WBC count in all the three routes of exposure. Histopathological evaluation of lung, liver, and spleen also showed that the damage was more in the p.c. route and severity of lesions was dependent on the dose of exposure. The most affected organ was liver by all the three routes. LD50 was also determined in male rats and it was found to be 2.4, 2.4, and 3.4 mg/kg through p.c., p.o. and s.c. routes respectively. Since skin contains maximum number of metabolically active and rapidly dividing cells, differential metabolism of SM cannot be ruled out. Probably, this is the first report of a chemical showing more toxicity through p.c. route compared to s.c. route.