CD40 is a membrane bound receptor molecule which is present on macrophages, B-cells, dendritic cells, T-cells and endothelial cells (EC) and plays a critical role in the development of atherosclerosis. Interestingly, our in vivo studies indicate that CD40 is usually not expressed in arterial but in venous EC. This asymmetric arteriovenous expression pattern does not reflect an imprinted EC phenotype as it is lost upon in vitro culture conditions. Nevertheless, this observation raises the question how endothelial CD40 could be involved in the development of atherosclerosis if it is usually not present in arteries. Delineating microenvironmental parameters potentially affecting CD40 expression we found that continued exposure to cyclic stretch as well as interaction with smooth muscle cells (SMC) down-regulates CD40 expression in EC. Accordingly, we analyzed CD40 expression in stretch stimulated EC interacting with SMC via cell-cell contacts or paracrine communication. We show that cyclic stretch induced CD40 downregulation in EC is reversed by direct but not paracrine contact to SMC. Furthermore, protein array studies identified TGF-b1 as well as its receptors to be responsible for SMC dependent reversed CD40 regulation. These results suggest a critical role for SMC in the regulation of arterial endothelial cell CD40 expression and may shed light into the complex regulatory mechanisms involved in atherosclerosis.