Respiratory syncytial virus (RSV) infection is a potent stimulus for airway epithelial expression of matrix metalloproteinase (MMP)-9. MMP-9 activity in vivo is a predictor of disease severity in children with RSV-induced respiratory failure. Human airway epithelial cells were infected with RSV A2 strain and analysed for MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 (a natural inhibitor of MMP-9) release. In addition, endotracheal samples from children with RSV-RF and controls (non-RSV pneumonia and nonlung disease controls) were analysed for MMP-9, TIMP-1, human neutrophil elastase and myeloperoxidase activity. RSV infection of airway epithelia was sufficient to rapidly induce MMP-9 transcription and protein release. Pulmonary MMP-9 activity peaked at 48 h in infants with RSV-induced respiratory failure. In the RSV group, MMP-9 activity and MMP-9/TIMP-1 ratio imbalance predicted higher oxygen requirement and worse paediatric risk of mortality scores. The highest levels of human neutrophil elastase and myeloperoxidase activity were measured in the RSV cohort; however, unlike MMP-9, these neutrophil markers failed to predict disease severity. These results support the hypothesis that RSV is a potent stimulus for MMP-9 expression and release from human airway epithelium, and that MMP-9 is an important biomarker of disease severity in mechanically ventilated children with RSV lung infection.