The cellular components of blood play a significant role in the breakdown of fibrin, with specific cellular adhesive processes allowing for accumulation of neutrophils within the fibrin clot. Fibrinolysis by elastase and cathepsin G, enzymes present within the azurophilic granules of the neutrophil, has previously been shown. Recent studies have demonstrated neutrophil-mediated fibrinogenolysis by a membrane-associated protease which suggests that proteases connected with the neutrophil membrane might also be capable of clot dissolution. Intact neutrophils were found to solubilize fibrin clots with the rate of fibrin solubilization being greater when the cells were incorporated into the clot than when the cells were added to preformed clots. Stimulation of intact neutrophils with phorbol ester upregulated this neutrophil-mediated fibrinolysis. Solubilization was detectable within 2 min of incubating the cells with fibrin clot, was always faster than that by neutrophil conditioned medium and lacked inhibition with lysosomal enzyme inhibitors. Neutrophil-mediated clot lysis was effected by membrane-associated serine proteases that migrated to apparent molecular weights of 501 kD, 398 kD, 316 kD, 245 kD and 209 kD on 3-13% SDS-PAGE. This degradation was distinct from that produced by plasmin, neutrophil lysosomal enzymes and purified human neutrophil elastase. The neutrophil membrane proteolytic systems were found to enhance the action of plasmin in clot solubilization. These results suggest neutrophil membrane proteolytic activity could assist thrombus dissolution and may be of particular value in assisting early clot dissolution by plasmin and when clot stabilization occurs through plasminogen activator inhibitor-1 (PAI-1) inhibition of plasminogen activation.