The majority of the animal models of Acute Lung Injury (ALI) are focused on the acute phase. This limits the studies of the mechanisms involved in later phases and the effects of long-term treatments. Thus, the goal of this study was to develop an experimental ALI model of aspiration pneumonia, in which diffuse alveolar damage continues for 72 h. Rats were intratracheally instilled with one dose of HCl (0.1 mol/L) followed by another instillation of one dose of LPS (0, 10, 20, 30 or 40 μg/g body weight [b.w.]) two hours later, which models aspiration of gastric contents that progresses to secondary lung injury from bacteria or bacterial products. The rats were sacrificed at 24, 48, and 72 h after the last instillation. The results showed that HCl and LPS at all doses caused activation of inflammatory responses, increased protein permeability and apoptosis in rat lungs at 24 h post-instillation. However, this lung damage was present at 72 h only in rats receiving HCl and LPS at the doses of 30 and 40 μg/g b.w. Mortality (50%) occurred in the first 48 h and only in the rats treated with HCl and LPS at the highest dose (40 μg/g b.w.). In conclusion, intratracheal instillation of HCl followed by LPS at the dose of 30 μg/g b.w. results in severe diffuse alveolar damage that continues at least 72 h. This rat model of aspiration pneumonia-induced ALI will be useful for testing long-term effect of new therapeutic strategies in ALI.