Swine barn air contains endotoxin and many other noxious agents. Single or multiple exposures to pig barn air induces lung inflammation and loss of lung function. However, we do not know the effect of exposure to pig barn air on inflammatory response in the lungs following a secondary infection. Therefore, we tested a hypothesis that single or multiple exposures to barn air will result in exaggerated lung inflammation in response to a secondary insult with Escherichia coli LPS (E. coli LPS). We exposed Sprague-Dawley rats to ambient (N = 12) or swine barn air (N = 24) for one or five days and then half (N = 6/group) of these rats received intravenous E. coli LPS challenge, observed for six hours and then euthanized to collect lung tissues for histology, immunohistochemistry and ELISA to assess lung inflammation. Compared to controls, histological signs of lung inflammation were evident in barn exposed rat lungs. Rats exposed to barn air for one or five days and challenged with E. coli LPS showed increased recruitment of granulocytes compared to those exposed only to the barn. Control, one and five day barn exposed rats that were challenged with E. coli LPS showed higher levels of IL-1beta in the lungs compared to respective groups not challenged with E. coli LPS. The levels of TNF-alpha in the lungs did not differ among any of the groups. Control rats without E. coli LPS challenge showed higher levels of TGF-beta2 compared to controls challenged with E. coli LPS. These results show that lungs of rats exposed to pig barn air retain the ability to respond to E. coli LPS challenge.