The binding of membrane-associated ephrinB ligands to corresponding EphB receptors in endothelial cells (ECs) is pivotal for the orchestrated development of the circulatory system. However, only little is known about its function in adult blood vessels.In this context, we observed that ephrinB1/ephrinB2-ligands are localized on the surface of ECs and their expression becomes up-regulated during inflammation. Consequently, we hypothesized that these ligands bind to EphB-receptors expressed in circulating leukocytes. To investigate functional consequences of such ligand-receptor interactions, ECs were exposed to EphB2 receptorbodies, EphB2-overexpressing myeloma cells or monocytes upon siRNA-mediated knockdown of ephrinB1 or ephrinB2. Based on this experimental setup, we analyzed the proinflammatory differentiation of the ECs and the transmigration of monocytes through the EC monolayer. Whereas forward signaling downstream of the EphB receptors promotes migration of monocytes, reverse ephrinB-signaling induces the release of von Willebrand factor and granulocyte-macrophage colony-stimulating factor, increases the expression of vascular cell adhesion molecule-1 and E-selectin in ECs and consequently stimulates monocyte attachment. Moreover, ephrinB1 and ephrinB2, which both are partially localized in the intercellular junctions of the ECs, are crucial for the transmigration of monocytes through the endothelial cell monolayer. Their activation by EphB2 is associated with a decline in the EC junctional integrity which is mediated by a src-dependent phosphorylation of CD31. Collectively, our data indicates for the first time that the ephrinB/EphB-system contributes to proinflammatory responses of ECs whereby ephrinB1 and B2 support adhesion and transmigration of EphB2-expressing monocytes and mediate the pro-inflammatory differentiation of ECs upon interaction with EphB2-expressing leukocytes.