Introduction: Exposure to mustard gas is known to result in severe lung damaging reactions. We have shown that instillation of liposomes containing reducing agents (N-acetylcysteine, NAC, glutathione, GSH) greatly attenuates acute lung injury caused by the mustard gas derivative 2-chloroethyl ethyl sulfide (CEES). However, pulmonary fibrosis remains a challenging long-term effect after exposure to mustard gas compounds. Therefore we conducted studies investigating the effect of liposomes containing various anti-oxidant agents on the long-term collagen deposition in the lung. Methods: Male Sprague-Dawley rats underwent airway instillation into the left lung of either CEES (2ml in 15% ethanol/PBS) or 15% ethanol/PBS (control) followed by intratracheal left lung administration of liposomes containing various anti-oxidants. Lungs were harvested at different time points for histological assessment of collagen deposition (Trichrome stain). For quantitative measurements of fibrosis, lung homogenates were assayed for hydroxyproline content as a surrogate of collagen. Results: During the first 24 hrs following CEES exposure of lungs, morpholocial evidence of acute lung injury was found. There was alveolar flooding, intraalveolar hemorrhage, interstitial and intraalveolar deposition of fibrin, and intense infiltrates of neutrophils. At 3 and 6 days, dense interstitial deposits of collagen were found. A collapse of alveolar structures, appearance of honeycombing, and dense collagen deposits indicative of pulmonary fibrosis were evident 3 weeks after CEES exposure. Subsequently, this time point was chosen for measurements of collagen amount. CEES-exposed rats treated with empty liposomes showed hydroxyproline levels of 10 mg/g dry weight. Administration of liposomes containing NAC, GSH, Vitamin E (a/g-tocopherol), NAC + GSH or a-lipoic acid failed to lower the degree of collagen deposition. In striking contrast, liposomes containing a combination of NAC + Vitamin E significantly decreased pulmonary fibrosis by more than 60%. Concluson: These results show for the first time that intratracheal instillation of CEES results in early histological alterations of the lung structure indicating acute lung injury. Further, we were able to show the usefulness of our model of CEES-exposure to mimic long-term effects of CEES. Collagen depositions were detectable 3-6 days after CEES exposure, while typical signs of pulmonary fibrosis (honeycombing) were seen 3 weeks after injury. Of the various anti-oxidant reagents administered within liposomes, only the combination of NAC and Vitamin E significantly prevented collagen deposition. This finding is in contrast to the protective effects of several anti-oxidants in the setting of acute lung injury occuring during the first 24 hrs after CEES exposure. Vitamin E is the primary in vivo anti-oxidant which plays an important role in scavenging oxygen radicals and stabilizing cell membranes. The combination with NAC, a reagent known to be a powerful and protective anti-oxidant, seems to enhance the anti-oxidant effects le ading to markedly less pulmonary fibrosis while administration of individual reducing reagents does not result in a similar protection.