Rationale: Elevated plasma and bronchoalveolar lavage fluid plasminogen activator inhibitor 1 (PAI-1) levels are associated with adverse clinical outcome in patients with pneumonia caused by Pseudomonas aeruginosa. However, whether PAI-1 plays a pathogenic role in the breakdown of the alveolar - capillary barrier caused by P aeruginosa is unknown. Objectives: The role of PAI-1 in pulmonary host defence and survival during P aeruginosa pneumonia in mice was tested. The in vitro mechanisms by which P aeruginosa causes PAI-1 gene and protein expression in lung endothelial and epithelial cells were also examined. Methods and results: PAI-1 null and wild-type mice that were pretreated with the PAI-1 inhibitor Tiplaxtinin had a significantly lower increase in lung vascular permeability than wild-type littermates after the airspace instillation of 1×107 colony-forming units (CFU) of P aeruginosa bacteria. Furthermore, P aeruginosa in vitro induced the expression of the PAI-1 gene and protein in a TLR4/p38/RhoA/NF-κB (Toll-like receptor 4/p38/RhoA/nuclear factor-κB) manner in lung endothelial and alveolar epithelial cells. However, in vivo disruption of PAI-1 signalling was associated with higher mortality at 24 h (p<0.03) and higher bacterial burden in the lungs secondary to decreased neutrophil migration into the distal airspace in response to P aeruginosa. Conclusions: The results indicate that PAI-1 is a critical mediator that controls the development of the early lung inflammation that is required for the activation of the later innate immune response necessary for the eradication of P aeruginosa from the distal airspaces of the lung.