Elastases in cystic fibrosis (CF) pulmonary fluids damage lung tissue and perpetuate cycles of infection, inflammation and injury. Elastases from three different sources may be present in CF airways: neutrophils, macrophages and Pseudomonas. We measured how well the cephalosporin-based antielastase L-658,758 blocks the activity of human neutrophil elastase (NE), human proteinase-3, human macrophage metalloelastase, mouse macrophage metalloelastase and Pseudomonas aeruginosa elastase. We also examined the ability of L-658,758 to block elastases in CF sputum in vitro. Sputum samples from adult CF patients were fractionated to obtain the aqueous sol phase. These were then studied individually or pooled. Elastinolytic activity, which ranged from 3.2 microg elastin degraded/ml sol/min to 26.3 microg elastin degraded/ml sol/min, was measurable in every individual sol sample and in the pooled sol. L-658,758 effectively inhibited elastinolysis by NE, proteinase-3 and the pooled sol but did not inhibit the activity of the metalloelastases, human and mouse macrophage metalloelastase and Pseudomonas elastase. Secretory leukoprotease inhibitor, which inhibited NE but did not inhibit proteinase-3, blocked 90% of sol elastinolytic activity; this suggests that the majority of this activity in the pooled sol derived from NE. L-658,758 was an effective inhibitor of sol elastase, blocking more than 97% of elastinolytic activity in the individual sol samples. We conclude that L-658,758 is an effective inhibitor of NE, proteinase-3 and CF sputum sol elastase.