In acute respiratory distress syndrome patients, protective ventilation strategies reduce mortality and proinflammatory mediator levels. It has been suggested that some of the side effects of mechanical ventilation are caused by the excessive release of mediators capable of causing pulmonary inflammation and tissue destruction (biotrauma). Selective inhibition of this process might be used to minimize the side effects of artificial mechanical ventilation. This study was designed to identify the cell types and specific signaling mechanisms that are activated by ventilation with increased pressure/volume (overventilation). In isolated perfused mouse lungs, overventilation caused nuclear translocation of nuclear factor-kappaB (NF-kappaB) and enhanced expression of interleukin-6 mRNA in alveolar macrophages and alveolar epithelial type II cells. The phosphoinositide 3-OH kinase inhibitor Ly294002 prevented nuclear translocation of NF-kappaB and the subsequent release of interleukin-6 and macrophage inflammatory protein-2alpha in overventilated but not in endotoxic lungs. Similar results were obtained in rats in vivo, where Ly294002 prevented NF-kappaB activation by overventilation but not by endotoxin. These findings show that alveolar macrophages and alveolar epithelial type II cells contribute to the ventilation-induced release of proinflammatory mediators and that selective inhibition of this process is possible without inhibiting the activation of NF-kappaB by endotoxin.