Matrix metalloproteinase-9 (MMP-9) is released by neutrophils at the sites of acute inflammation. This enzyme modulates matrix turnover and inflammatory response and its activity has been found increased after ventilator-induced lung injury. To clarify the role of MMP-9, mice lacking this enzyme and their wildtype counterparts were ventilated for two hours with high or low peak inspiratory pressures (25 and 15 cmH₂0, respectively). Lung injury was evaluated by gas exchange, respiratory mechanics, wet-to-dry weight ratio and histologic analysis. The activity of MMP-9 and levels of interleukin-1, interleukin-4 and macrophage inflammatory protein (MIP-2) were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Cell count and myeloperoxidase activity were measured in BALF. There were no differences between wildtype and Mmp-9^-/- animals after low-pressure ventilation. After high pressure ventilation, wildtype mice exhibited an increase in MMP-9 in tissue and BALF. Mice lacking MMP-9 developed more severe lung injury than wildtype mice, in terms of impaired oxygenation and lung mechanics, and higher damage in the histological study. These effects correlated with an increase in both cell count and myeloperoxidase activity in the BALF, suggesting an increased neutrophilic influx in response to ventilation. An increase in IL-1 and IL-4 in the BALF only in knockout mice could be responsible for the differences. There were no differences between genotypes in MMP-2, MMP-8 or tissue inhibitors of metalloproteinases. These results show that MMP-9 protects against ventilator-induced lung injury by decreasing alveolar neutrophilic infiltration, probably by modulation of the cytokine response in the air spaces.