The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1beta, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.