Permissive hypercapnia because of reduced tidal volume is associated with improved survival in lung injury, whereas therapeutic hypercapnia-deliberate elevation of arterial Pco2-protects against in vivo reperfusion injury and injury produced by severe lung stretch. No published studies to date have examined the effects of CO2 on in vivo models of neonatal lung injury. We used an established in vivo rabbit model of surfactant depletion to investigate whether therapeutic hypercapnia would improve oxygenation and protect against ventilator-induced lung injury. Animals were randomized to injurious (tidal volume, 12 mL/kg; positive end-expiratory pressure, 0 cm H2O) or protective ventilatory strategy (tidal volume, 5 mL/kg; positive end-expiratory pressure, 12.5 cm H2O), and to receive either control conditions or therapeutic hypercapnia (fraction of inspired CO2, 0.12). Oxygenation (alveolar-arterial O2 difference, arterial Po2), lung injury (alveolar-capillary protein leak, impairment of static compliance), and selected bronchoalveolar lavage and plasma cytokines (IL-8, growth-related oncogene, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha) were measured. Injurious ventilation resulted in a large alveolar-arterial O2 gradient, elevated peak airway pressure, increased protein leak, and impaired lung compliance. Therapeutic hypercapnia did not affect any of these outcomes. Tumor necrosis factor-alpha was not increased by mechanical stretch in any of the groups. Therapeutic hypercapnia abolished the stretch-induced increase in bronchoalveolar lavage monocyte chemoattractant protein-1, but did not affect any of the other mediators studied. Therapeutic hypercapnia may attenuate the impairment in oxygenation and inhibit certain cytokines. Because hypercapnia inhibits certain cytokines but does not alter lung injury, the pathogenic role of these cytokines in lung injury is questionable.