Sequestration of activated polymorphonuclear leukocytes (PMN) within the lung microcirculation may contribute to pulmonary vascular injury following trauma, sepsis, or disseminated intravascular coagulation. In this study cultured rat endothelial cells were utilized to evaluate the effect of PMN activation on endothelial cell attachment. The concept that disruption of the extracellular fibronectin matrix is associated with altered endothelial cell adhesion was also tested. Rat endothelial cells were grown in culture and identified by morphological techniques as well as immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with 51Cr in order to establish a cell injury assay based on release of free 51Cr or cell-associated 51Cr. PMN activation was verified microscopically and by chemiluminescence activity following phorbol myristate acetate (PMA) or opsonized zymosan exposure. Following incubation with PMA, the leukocytes aggregated, chemiluminesced vigorously, and caused endothelial cell injury and detachment as determined by release of 51Cr-labeled endothelial cells. PMNs exposed to serum-treated zymosan exhibited a more modest chemiluminescence burst which was consistent with their decreased activity to injure the endothelial monolayer. With PMA activation the degree of endothelial detachment from the monolayer increased as a function of time with a plateau observed by 3 hr. Microscopic immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin after incubation with PMA-activated neutrophils in association with endothelial cell disadhesion. Thus, exposure of activated rat PMN to rat endothelial cells in culture induces endothelial damage and an associated disruption of the fibronectin matrix which may contribute to endothelial cell detachment.