This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V_T) might affect the composition and/or structure of the glycosaminoglycans (GAGs) components of pulmonary extracellular proteoglycans (PGs). Experiments were performed in 30 healthy rats: 1) anesthetized and immediately sacrificed (Controls, C-0); 2) anesthetized and spontaneously breathing for 4 hours (C-4h); 3) anesthetized, paralyzed and mechanically ventilated for 4 hours with air at 0 cmH₂O end-expiratory pressure with V_T of 8 ml·Kg^-1 (MV-1), 16 ml·Kg^-1 (MV-2), 24 ml·Kg^-1 (MV-3) or 32 ml·Kg^-1 (MV-4), adjusting respiratory rates to maintain a minute ventilation of 270 ml·min^-1 . Compared to C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, mechanical ventilation induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bond GAGs and the wash-out of loosely bond or fragmented GAGs progressively increased with increasing V_T and was associated with an increased expression of local (MMP-2) and systemic (MMP-9) activated metalloproteases. We conclude that: 1) mechanical ventilation even at "physiological" low V_T, severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.