Abstract After injury and vascular replacement, endothelial cell recovery is limited and could lead to thrombosis. Seeding small diameter vascular prosthesis with endothelial cells has been proposed to fulfil cell lining and improve surface hemocompatibility. However, detachment of seeded cells occurs following implantation. Previous in vitro studies have looked at the fluid shear stress as a major cause of cell detachment. To our knowledge, the role of erythrocyte collisions has not been investigated. The present in vitro study aims at investigating whether endothelial cell adhesion depends on (i) the presence of erythrocytes in flow and (ii) the latent culture period (1, 24 and 48 h) between seeding and exposure to flow. Endothelial cells were exposed to culture media containing different erythrocyte concentrations using a steady laminar flow of 1350 ml min -1 in a parallel plate flow chamber. Endothelial cell morphology in dynamic conditions was quantified and compared to that in static conditions. The projected area of cells were mostly found smaller under dynamic than static conditions, particularly at a wall shear stress of 23 dyn cm -2. Cells from the 1 h latent culture period were oriented parallel to the flow axis and were more elongated than under static conditions. Conversely, endothelial cell shape was slightly modified when either the latent period or the wall shear stress was increased. Disparate orientation was observed on confluent endothelial cells (24–48 h latent period) exposed to shear stress with or without erythrocytes. Increasing fluid viscous forces due to erythrocytes play a critical role on the behaviour of freshly seeded endothelial cells upon exposure to blood flow.