We studied the interaction of [125I]fibronectin with human umbilical vein endothelial cells. Endothelial cell monolayers cross-linked [125I]fibronectin which had been preadsorbed to gelatin-coated dishes. The cross-linking of the substrate-immobilized [125I]fibronectin was mediated by cell-associated tissue transglutaminase and occurred more rapidly during the first 30 min after endothelial cell seeding but also continued for several hours after the cells were fully spread. The processing of the [125I]fibronectin was associated with the basolateral surface of the endothelial cell, as demonstrated by the finding that cross-linking did not occur when [125I]fibronectin was presented to the apical surface of confluent monolayers. Transglutaminase activity was not necessary for attachment and spreading of HUVEC on a fibronectin/gelatin matrix. The presence of a nonpeptidyl transglutaminase inactivator rendered the cells more susceptible to detachment by trypsin and destabilized the association of fibronectin with the subendothelial extracellular matrix. Thus, endothelial cells process fibronectin into cross-linked multimers due to the expression of tissue transglutaminase at the basal surface of the cell. This process may serve to stabilize the extracellular matrix and to firmly anchor the cells to the basement membrane.