Most clinical strains of Pseudomonas aeruginosa produce elastase, a zinc metalloprotease that is implicated in the pathogenesis of infections related to these organisms. To better understand the physiologic role of this protease in the regulation of airway permeability, we developed a panel of specific monoclonal antibodies (mAb) against purified Pseudomonas elastase (PE) that do not react with either neutrophil elastase or porcine pancreatic elastase. These mAbs were used in a competitive enzyme-linked immunosorbent assay to determine the concentrations of PE in sputum samples from patients with pulmonary infections. Sputum from patients infected with P. aeruginosa showed a varying amount of PE, whereas others indicated no signals. We also found that the mAbs blocked the effect of PE on epithelial barrier function in vitro on the basis of measurement of transmonolayer electrical resistance of polarized epithelial cells as an index of paracellular permeability.