The combined effect of mechanical ventilation and fluid load on pulmonary glycasaminoglycans (GAGs) was studied in anaesthetized rats ((BW 290 ± 21.8 (SE) g) mechanically ventilated for 4 h: (a) at low (∼7.5 ml kg−1) or high (∼23 ml kg−1) tidal volume (VT) and zero alveolar pressure; (b) at low or high VT at 5 cmH2O positive end-expiratory pressure (PEEP); (c) with or without 7 ml kg−1 h−1 intravenous infusion of Phosphate Buffer Solution (PBS). Compared to spontaneous breathing, GAGs extractability decreased by 52.1 ± 1.5% and 42.2 ± 7.3% in not-infused lungs mechanically ventilated at low VT or at high VT and PEEP, respectively. In contrast, in infused lungs, GAGs extractability increased by 56.1 ± 4.0% in spontaneous ventilation and PEEP and up to 81.1% in all mechanically ventilated lungs, except at low VT without PEEP. In the absence of an inflammatory process, these results suggest that PEEP was protective at low but not at high VT when alveolar structures experience exceedingly high stresses. When combined to mechanical ventilation, fluid load might exacerbate edema development and lung injury.