N-acetylcysteine (NAC) suppresses the generation of reactive oxygen species (ROS) that are implicated in ventilator-induced lung injury (VILI). We thus hypothesised that NAC attenuates VILI. VILI was induced by mechanical ventilation with a tidal volume (Vt) of 15mlkg(-1) in isolated and perfused rat lung. NAC was administered in the perfusate prior to the onset of mechanical ventilation. A group ventilated with low Vt of 5mlkg(-1) served as control. Haemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined upon completion of the mechanical ventilation. There was an increase in lung permeability and lung weight gain after mechanical ventilation with high Vt, compared to low Vt. The levels of inflammatory cytokines including interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and macrophage inflammatory protein-2 (MIP-2) increased in lung lavage fluids; the concentrations of H(2)O(2) were higher in lung lavage fluids, and the expression of myeloperoxidase (MPO), JNK, P38, pAKT and caspase-3 in lung tissue was greater in the high Vt than in the low Vt group. The concentrations of glutathione (GSH) in lung tissue were higher in low Vt than those in high Vt. The administration of NAC increased GSH, attenuated ROS, cytokines, MPO, JNK, pAKT and caspase-3 and lung permeability associated with decreased activation of nuclear factor-κB. VILI is associated with inflammatory responses including the generation of ROS, cytokines and the activation of mitogen-activated protein kinase cascade. The administration of NAC attenuates the inflammatory responses, apoptosis and VILI in the isolated, perfused rat lung model.