To investigate the changes in pulmonary transforming growth factor-beta1 (TGF-beta1)/smad2 signaling pathway in pulmonary injury as a result of hemorrhagic shock followed by lipopolysaccharide challenge. Twenty-four Sprague-Dawley (SD) rats were randomly assigned to the following two groups: sham operation group (sham group, surgery, no hemorrhage and no resuscitation), and two-hit model group (HS group), each n=12. Three-phased uncontrolled hemorrhagic shock model was reproduced in rats. Hemorrhagic shock phase I began with blood withdrawal over 15 minutes, i.e. animals were subjected to massive hemorrhage [mean arterial pressure (MAP)=35-40 mm Hg (1 mm Hg=0.133 kPa) for 60 minutes], followed by intratracheal lipopolysaccharide 2 mg/kg (two-hit model). Ninety minutes after blood shedding, resuscitation phase II of 60 minutes began with hemostasis, return of all the blood initially shed, plus fluids. Observation phase III was 210 minutes. After phase III, blood gas analysis with carotid artery blood was performed. Lung tissue was sampled to measure values of wet-to-dry lung weight (W/D) ratio and pulmonary microvascular permeability. Immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) were used to assess the expression of TGF-beta1 protein and mRNA, and the protein content of the smad2 was determined by Western blotting. Compared with sham group, MAP was significantly lowered after 60 minutes in phase I, and lactic acid content was increased significantly, while partial pressure of oxygen in artery (PaO2), blood pH, HCO(-)3, arterial oxygen saturation (SaO2) and negative base excess (BE) showed a significant decrease in HS group. Concomitantly, values of pulmonary microvascular permeability and W/D ratio were significantly increased in HS group (all P<0.01). In sham group, weak TGF-beta1 staining was detected in the alveolar epithelial cells. However, intense positive immunostaining for TGF-beta1 was observed in alveolar epithelial cells, pulmonary interstitial inflammatory cell as well as macrophage cells of alveolar space of the HS group. Lung tissue in HS group demonstrated a marked increase in TGF-beta1 mRNA and smad2 protein expression in the lung tissue compared with those of sham group (all P<0.01). The expression of TGF-beta1/smad2 signaling pathway may play an important role in regulation of pulmonary permeability and development of pulmonary edema in acute lung injury induced by uncontrolled hemorrhagic shock followed by lipopolysaccharide challenge.