Rationale: Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress Objective: To define the relative contributions of alveolar overdistension and cyclic recruitment and "collapse" of unstable lung units to membrane wounding of alveolar epithelial cells. Methods: We measured the interactive effects of tidal volume (VT), transpulmonary pressure (PTP) and of airspace liquid on the number of alveolar epithelial cells with plasma membrane wounds in ex vivo mechanically ventilated rat lungs. Plasma membrane integrity was assessed by Propidium Iodide (PI) exclusion in confocal images of subpleural alveoli. Main Results: Cyclic inflations of normal lungs from zero end-expiratory pressure (ZEEP) to 40 cm H2O produced VT's of 56.9±3.1cc/kg and were associated with 0.12±0.12 PI positive cells per alveolus. A preceding tracheal instillation of normal saline (3ml) reduced VT to 49.1±6cc/kg, but was associated with a significantly greater number of wounded alveolar epithelial cells (0.52±0.16 cells per alveolus; p<0.01). Mechanical ventilation of completely saline filled lungs with saline (VT=52cc/kg) to pressures between 10 and 15 cm H2O was associated with the least number of wounded epithelial cells (0.02±0.02 cells per alveolus; p<0.01). In mechanically ventilated, partially saline filled lungs the number of wounded cells increased substantially with VT, but once VT was accounted for, wounding was independent of maximal PTP. Conclusions: Interfacial stress associated with the generation and destruction of liquid bridges in airspaces is the primary biophysical cell injury mechanism in mechanically ventilated lungs.