We previously reported the expression of the two-pore-domain K channel TREK-1 in lung epithelial cells and proposed a role for this channel in the regulation of alveolar epithelial cytokine secretion. In this study, we focused on investigating the role of TREK-1 in vivo in the development of hyperoxia-induced lung injury. Laboratory animal experiments. University research laboratory. Wild-type and TREK-1-deficient mice. Mice were anesthetized and exposed to 1) room air, no mechanical ventilation, 2) 95% hyperoxia for 24 hours, and 3) 95% hyperoxia for 24 hours followed by mechanical ventilation for 4 hours. Hyperoxia exposure accentuated lung injury in TREK-1-deficient mice but not controls, resulting in increase in lung injury scores, bronchoalveolar lavage fluid cell numbers, and cellular apoptosis and a decrease in quasi-static lung compliance. Exposure to a combination of hyperoxia and injurious mechanical ventilation resulted in further morphological lung damage and increased lung injury scores and bronchoalveolar lavage fluid cell numbers in control but not TREK-1-deficient mice. At baseline and after hyperoxia exposure, bronchoalveolar lavage cytokine levels were unchanged in TREK-1-deficient mice compared with controls. Exposure to hyperoxia and mechanical ventilation resulted in an increase in bronchoalveolar lavage interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α levels in both mouse types, but the increase in interleukin-6 and monocyte chemotactic protein-1 levels was less prominent in TREK-1-deficient mice than in controls. Lung tissue macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin-1β gene expression was not altered by hyperoxia in TREK-1-deficient mice compared with controls. Furthermore, we show for the first time TREK-1 expression on alveolar macrophages and unimpaired tumor necrosis factor-α secretion from TREK-1-deficient macrophages. TREK-1 deficiency resulted in increased sensitivity of lungs to hyperoxia, but this effect is less prominent if overwhelming injury is induced by the combination of hyperoxia and injurious mechanical ventilation. TREK-1 may constitute a new potential target for the development of novel treatment strategies against hyperoxia-induced lung injury.