Respiratory tract lesions induced by the chemical warfare agent sulfur mustard (SM) are characterized by epithelial damages associated with inflammatory cell infiltration. Here, we evaluated the imbalance between gelatinase and tissue inhibitors of MMPs (TIMPs) and we tested pretreatment with the protease-inhibitor doxycycline. Guinea pigs were intoxicated intra-tracheally with SM and evaluated 24 hours after exposure. MMP gelatinase activity of bronchial lavage (BL) fluid from SM-exposed guinea pigs was high compared to controls, as shown by both zymography and biotinylated substrate degradation, whereas TIMP-1 and -2 levels by immunoblotting were similar. Extensive areas of lysis were evidenced by in situ zymography, indicating imbalance between gelatinases and inhibitors towards net proteolytic activity. Doxycycline pretreatment resulted in i) decreased gelatinase activity (zymography, free gelatinase activity assay, and in situ zymography); ii) decreased inflammation (BL fluid cellularity and protein level); and iii) dramatic decrease in histological epithelial lesions. Our results suggest inadequate level of TIMP to counteract increased gelatinase activity, and further support a role for MMP gelatinases in SM-induced respiratory lesions. They also suggest that doxycycline may hold promise as a therapeutic tool.