Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)- causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF- have not been defined. Because TNF- can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF- (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface ₅₁-Fn integrins with the Fn fibers in focal contacts was also observed. TNF- treatment of endothelial monolayers with matrices prelabeled with ¹²⁵I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF- exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF- increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF--induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF- appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased ₅₁-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF- exposure.