Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)- causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF- (200 U/ml) for 18 h. Western blot analysis indicated that TNF- decreased the amount of monomeric FN detected under reducing conditions. Analysis of ¹²⁵I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF- also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF--induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF- exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.