Neutrophil elastase (NE), a serine protease present in high concentrations in the airways of cystic fibrosis patients, injures the airway epithelium. We examined the epithelial response to NE-mediated proteolytic injury. We have previously reported that NE treatment of airway epithelial cells causes a marked decrease in epithelial DNA synthesis and proliferation. We hypothesized that NE inhibits DNA synthesis by arresting cell cycle progression. Progression through the cell cycle is positively regulated by cyclin complexes and negatively regulated by cyclin-dependent kinase inhibitors (CKI). To test whether NE arrests cell cycle progression, we treated normal human bronchial epithelial (NHBE) cells with NE (50 nM) or control vehicle for 24 h and assessed the effect of treatment on the cell cycle by flow cytometry. NE treatment resulted in G(1) arrest. Arrest in G(1) phase may be the result of CKI inhibition of the cyclin E complex; therefore, we evaluated whether NE upregulated CKI expression and/or affected the interaction of CKIs with the cyclin E complex. Following NE or control vehicle treatment, expression of p27(Kip1), a member of the Cip/Kip family, was evaluated. NE increased p27(Kip1) gene and protein expression. NE increased the coimmunoprecipitation of p27(Kip1) with cyclin E complex, suggesting that p27(Kip1) inhibited cyclin E complex activity. Our results demonstrate that p27 is regulated by NE and is critical for NE-induced cell cycle arrest.