Polymorphonuclear leukocytes (PMN) contribute to increased pulmonary vascular permeability in inflammatory lung injury, but the mechanism of their action is complex. In the present study we examined possible effects of PMN-derived proteases on the permeability of pulmonary endothelial cell monolayers grown on polycarbonate filter membranes and exposed continuously to a hydrostatic pressure of 10 cm H2O. Cell- and serum free PMN-supernatants (human PMN, stimulated with 30 ng/ml phorbol-myristate acetate for 30 min, presence of catalase, were centrifuged, the supernatants were passed through a 0.45 micron filter) dose-dependently (calculated PMN: endothelial cell ratio of 2:1 and more) increased hydraulic conductivity of endothelial cell monolayers ten- to twentyfold within 20-70 min. At the same time the dextran reflection coefficient decreased from 0.8 to 0.1. Phase contrast and scanning electronmicroscopy showed a widening of intercellular gaps. The effects of the postsecretory PMN-supernatant were blocked dose-dependently by inhibitors of human neutrophil elastase (HNE) but not of cathepsin G. On quantitative grounds highly purified HNE was similarly active as postsecretory PMN supernatant. The effects of HNE were inhibited by pretreatment with eglin-c or heat, but not with heparin. The data suggest that HNE is an effective and sufficient neutrophil-derived mediator to increase endothelial permeability. HNE appears to act primarily enzymatically and not as a cationic protein.