These studies evaluated whether the increased adherence of neutrophils to endothelium after exposure to lipopolysaccharide (LPS) endotoxin is primarily an effect on neutrophils or on endothelial cells. The studies demonstrate that preincubation of monolayers of human umbilical vein endothelial cells with LPS has a significantly greater effect on neutrophil adherence to endothelium than does preincubation of neutrophils with LPS (P less than 0.001 for each amount of LPS). Although the effect was small compared with incubation of endothelial cells with LPS, incubation of neutrophils with LPS did significantly increase their subsequent adherence to endothelial cells compared with controls (P less than 0.05). LPS was not toxic to either endothelial cells or neutrophils, as measured by the release of lactate dehydrogenase. Preincubation of endothelial cells with LPS at a concentration of 1.0 to 10 micrograms/ml maximally increased their ability to bind to neutrophils, and this effect was maximally expressed after 4 hours of exposure to LPS. In the assay, neutrophil binding to LPS-stimulated endothelial cells was rapid and did not increase after 30 minutes of coculture of neutrophils and endothelium. Morphologic studies demonstrated that LPS opened cell-to-cell junctions between endothelial cells. Neutrophils that attached to these monolayers of LPS-stimulated endothelial cells bound, primarily, to the margins of the endothelial cells and not to the underlying tissue culture dishes, which were exposed after incubation with LPS. These observations suggest that LPS increases neutrophil adherence primarily, but not solely, via an effect on endothelial cells.