Animal model is of importance to further elucidate the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). We envisioned a possibility that there might be the differences in lipopolysaccharide (LPS)-induced acute lung inflammation by the trans-oral and trans-tracheal intratracheal instillations. We compared the LPS-induced early inflammatory responses by these two methods. The evaluative system included bronchoalveolar lavage (BAL) fluid biochemical analysis and differential cell counting, lung wet/dry weight ratio and lung histology. In vitro studies were performed on human bronchial epithelial cell line NCI-H292 and alveolar Type II epithelial cell line A549 stimulated with LPS. Both interleukin (IL)-8 release in the BAL fluid and IL-8 secretions from NCI-H292 and A549 cells were measured. We found that the trans-tracheal intratracheal instillation promoted the LPS-induced cell injury, neutrophil infiltration, and pulmonary edema compared to the trans-oral one. The LPS-induced pathological changes by the trans-oral intratracheal instillation were characterized by pulmonary interstitial edema, but the trans-tracheal intratracheal instillation was exudative pulmonary edema. More IL-8 is produced from A549 cells than from NCI-H292 cells under the treatment of LPS. The increased IL-8 release in the BAL fluid and enhanced inflammatory responses caused by LPS may be due to more LPS delivered into the alveolar spaces by the trans-tracheal intratracheal instillation compared to the trans-oral one. The trans-tracheal intratracheal instillation is proved to be more suitable to establish the murine model of ALI than the trans-oral one and helpful to further elucidate the pathogenesis of ALI/ARDS. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.