It has been reported that cultured endothelial cells become procoagulant when exposed to endotoxin. This prompted us to investigate whether human endothelial cells treated with endotoxin could promote the generation of fibrin when exposed to human flowing blood. For this purpose we used a parallel-plate perfusion chamber in which confluent cultured endothelial cells from human umbilical veins were exposed for five minutes to directly drawn human nonanticoagulated blood, at wall shear rates of 100, 650, and 2600 sec-1. Fibrin deposition was assessed by morphometry. No fibrin deposition occurred on normal endothelial cells. In contrast, cells incubated with endotoxin for 4 or 18 hours induced fibrin deposition, but only at a shear rate of 100 sec-1. Since some extracellular matrix was exposed between the cells, we investigated whether extracellular matrix played a role in fibrin formation. When the endothelial cells incubated or not with endotoxin were removed by EDTA, the exposed extracellular matrix perfused with blood at 100 sec-1 supported platelet and fibrin deposition in both cases. This suggests that the effect of endotoxin on endothelial cells was not due to extracellular matrix alteration but only to cellular activation or secretion of procoagulant substances. We conclude that human endothelial cells treated with endotoxin can trigger fibrin formation and deposition at their surface when exposed to flowing blood at low shear rate.