The human acute respiratory distress syndrome (ARDS) is a severe pulmonary complication with high mortality rates. To support their vital functions, patients suffering from ARDS are mechanically ventilated. Recently it was shown that low tidal volume ventilation reduces mortality and pro-inflammatory mediator release in these patients, suggesting biotrauma as a side effect of mechanical ventilation. Because the application of exogenous surfactant has been proposed as a treatment for ARDS, we investigated the effect of surfactant on ventilation-induced release of tumor necrosis factor (TNF), interleukin-6 (IL-6) and 6-keto-PGF1α (the stable metabolite of prostacyclin) in isolated perfused mouse lungs ventilated with high end-inspiratory pressures. Instillation of 100 mg/kg surfactant into the lungs was well tolerated and improved tidal volume, pulmonary compliance and alveolar expansion. Exogenous surfactant increased the ventilation-induced liberation of TNF and IL-6 into the perfusate, but had no effect on the release of 6-keto-PGF1α. The surfactant preparation used reduced baseline TNF production by murine alveolar macrophages, indicating that the exaggeration of ventilation-induced TNF release cannot be explained by a direct effect of surfactant on these cells. We hypothesize that ventilation-induced mediator release is explained by stretching of lung cells, which is reinforced by surfactant. The findings that in this model of ventilation-induced lung injury exogenous surfactant at the same time improved lung functions and enhanced mediator release suggest that surfactant treatment may prevent barotrauma and augment biotrauma.