CD40-CD40 ligand (CD154) interactions not only play a role in cell-mediated immune responses but also activate endothelial cells and promote inflammatory cell recruitment during the initiation and progression of atherosclerosis. Platelet-bound and shedded soluble CD154 (sCD154) seem to be intimately involved in different steps of the atherogenic cascade while endothelial cell CD40 is preferentially expressed at atherosclerosis predilection sites of the arterial tree. The aim of the present study was to determine the pathophysiological relevance of such platelet CD154 endothelial cell CD40 interactions in the early phase of atherosclerosis. Human umbilical vein endothelial cells maintained under static conditions revealed a transient increase in intracellular calcium in single endothelial cells interacting with myeloma cells stably expressing human CD154. A similar rise in intracellular calcium was observed in HUVECs superfused with recombinant soluble CD154 by using Fura-2 AM ratio imaging analysis. The CD154 expressing myeloma cells elicited the release and luminal attachment of ultra-large von Willebrand factor (vWF) fibers on the endothelial cell surface under static conditions as well as recombinant soluble CD154 under low shear stress conditions in a concentration dependent manner. This release of vWF into the conditioned medium of the HUVECs was confirmed by using a specific ELISA. CD154 stimulation of human endothelial cells thus results in the calcium-dependent release of vWF from the Weibel-Palade bodies and the formation of ultra-large vWF fibres on the luminal endothelial cell surface under flow. This increased adhesiveness of the endothelial cells may subsequently lead to an enhanced recruitment of platelets, reinforcing their interaction with the endothelial cells and possibly facilitating an enhanced diapedesis of leukocytes that may contribute to the initiation of atherosclerosis.