Fibronectin is found in a soluble form in plasma as well as in an insoluble form in tissues. It is produced by cultured endothelial cells and can be localized in vitro and in vivo between adjacent endothelial cells as well as underneath endothelial cells in association with their collagenous matrix, where it is believed to influence cell adhesion. Fibronectin is susceptible to proteolytic cleavage by enzymes released from activated leukocytes. In the present study, cultured rat endothelial cells developed a fibrillar fibronectin network in their extracellular matrices in addition to releasing soluble, intact fibronectin (440 kDa) into their culture medium. Exposure of monolayers of cultured endothelial cells to activated polymorphonuclear leukocytes (PMNs) results in disruption of the fibrillar matrix fibronectin, damage to the endothelial cell monolayer, and presence of fibronectin fragments in the culture medium. In addition, acute leukocyte activation and peripheral leukopenia in vivo as induced by the intravenous infusion of foreign test particles also resulted in the appearance of low-molecular-weight fibronectin fragments in plasma. In the in vivo studies, the appearance of fibronectin fragments preceded the release of intact 440-kDa fibronectin in the plasma after its acute depletion by particle injection. Thus, activated leukocytes, adherent to an endothelial surface in vitro and in vivo, may result in degradation of matrix fibronectin and the release of fibronectin fragments into the extracellular environment. In vivo fibronectin fragments in blood may serve as a stimulus for the subsequent synthesis and/or release of intact plasma fibronectin.