Human rhinovirus (HRV) infections up-regulate pro-inflammatory mediators and growth factors that are associated with exacerbations of inflammatory airway diseases, such as asthma and COPD. Matrix metalloproteinase (MMP)-9 has been shown to be increased in the airways of patients with asthma and COPD. In this study we sought, firstly, to determine whether HRV infection modulated the expression of MMP-9 and its highest affinity inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 and, secondly, to explore the mechanism by which this occurs. In vitro studies, using RT-PCR, ELISA, zymography and a fluorescent activity assay, demonstrated that MMP-9 mRNA, protein and activity were increased upon infection with HRV, while TIMP-1 mRNA and protein remained unchanged. These results were then verified in vivo, using nasal lavage samples obtained from subjects with confirmed rhinovirus infections. HRV infections have been shown to upregulate NF-B, and NF-B has also been reported to play a role in MMP-9 expression. We therefore investigated the role of NF-B in HRV-induced MMP-9 expression. Using two inhibitors of IB kinase , we observed a concentration-dependent decrease in HRV-induced MMP-9 expression. The role of NF-B in HRV-induced MMP-9 expression was further confirmed using MMP-9 promoter luciferase constructs, which demonstrated that an NF-B site at -620/-607 bp was necessary for HRV-induced MMP-9 expression. Electrophoretic mobility shift assays and supershift assays confirmed nuclear translocation and binding of p50/p65 NF-B subunits to a MMP-9 specific NF-B oligonucleotide. This increase in MMP-9 may be a mechanism by which rhinovirus infections contribute to airway inflammation and, potentially, to airway remodeling.