A growing body of evidence suggests that neutrophil-derived proteinases play a major role in lung tissue damage in cystic fibrosis (CF). Most previous studies have focused on serine proteinases such as neutrophil elastase, providing no information on the extent to which metalloproteinases participate in proteolytic processes in CF. To address this issue, we evaluated the contribution of one of the major neutrophil metalloproteinases, i.e., 95 kDa gelatinase (type IV collagenase), to the total gelatinolytic activity measured in sputum specimens from 27 patients with CF. Compared with asthmatic children (n = 9), CF patients had a 6.7 times greater level of total gelatinase activity in sputum revealed by zymography. The 95 kDa gelatinase was increased 3.7-fold in the CF subjects (2,441 +/- 411 [SEM] arbitrary units [AU] x 10(6) per ml of sputum versus 665 +/- 201 in asthmatics) and the 88-kDa active form 23.2-fold (2,272 +/- 372 AU x 10(6) per ml of sputum versus 98 +/- 43, respectively). Using radiolabeled 3H-gelatin as the substrate, we demonstrated uninhibited gelatinolytic activity in all CF patients; this activity was significantly correlated to disease severity as assessed by pulmonary function tests. Western blotting using anti-tissue inhibitor of metalloproteinase (anti-TIMP) and anti-95/88-kDa gelatinase antibodies demonstrated a more than 10-fold excess of 95/88 kDa gelatinase over TIMP. Bacterial proteinases from Pseudomonas aeruginosa were shown to contribute little to the gelatinolytic activity measured in sputum supernatants from patients with CF, although culture supernatants from various P. aeruginosa strains expressed gelatinolytic activity in vitro. Finally, lung damage, as assessed by increased type IV collagen degradation products in sputum, was significantly correlated to concentrations of active 88 kDa gelatinase. These data argue for a significant role of 95/88 kDa gelatinase in airway damage in CF.