We evaluated the roles of proteinase 3 (PR3) and human neutrophil elastase (HNE), two neutrophil serine proteinases in the mechanisms leading to airway inflammation and hypersecretion in cystic fibrosis (CF). Using specific enzyme-linked immunosorbent assay (ELISA), we found higher levels of PR3 than HNE in sputum from CF patients. Using two inhibitors, ICI (Imperial Chemical Industries) 200,355 (which inhibits both HNE and PR3) and secretory leukoproteinase inhibitor (SLPI) (which inhibits only HNE), we showed that PR3 was enzymatically active in sputum, and its activity, as assessed by SLPI-resistant serine proteinase activity, correlated highly with its antigenic concentration measured by ELISA. Interestingly, sputum pellet-associated serine proteinase activity was mostly due to HNE. PR3 purified from neutrophil azurophil granules triggered airway gland secretion, as measured by the release of radiolabeled molecules from cultured bovine tracheal serous cells pulse-labeled with Na₂ ³⁵SO₄. This secretory activity was inhibited by ICI 200,355. PR3 concentration in CF sputum was highly correlated with taurine concentration, a reliable marker of airway inflammation and respiratory scores (e.g., FEV₁%), whereas no significant correlation was observed with HNE. We verified that Pseudomonas aeruginosa proteinases did not interfere with the assessment of PR3 and HNE. Indeed, the PR3/HNE ratio was greatest in patients chronically infected by P. aeruginosa. We suggest that PR3 may play a role in the hypersecretory process that is characteristic of CF.