The ligand ephrin A1 is more often discussed to play a role in the development of the atherosclerotic plaque and in this context especially in the monocyte adhesion to endothelial cells. As tumor necrosis factor-α (TNF-α) is known to induce monocyte adhesion to endothelium and ephrin A1 expression, the present study focuses on the involvement of ephrin A1 in TNF-α-mediated monocyte adhesion. The analysis of different members of the Eph/ephrin system in TNF-α-treated human umbilical vein endothelial cells (HUVEC) revealed that especially ephrinA1 was found to be highly regulated by TNF-α compared to other members of the Eph family. This effect is also present in arterial endothelial cells from the umbilical artery and from the coronary artery. This regulation is dependent on NFκB-activation as shown by the expression of a constitutive-active IκB-mutant. By using siRNA-mediated silencing and adenoviral overexpression of ephrinA1 in HUVEC, the involvement of ephrinA1 in the TNF-α triggered monocyte adhesion to endothelial cells could be demonstrated. In addition, these results could be verified by quantitative adhesion measurement using atomic force microscopy-based single-cell force spectroscopy and under flow conditions. Furthermore, this effect is mediated via the EphA4 receptor. EphrinA1 does not influence the mRNA or protein expression of the adhesion receptors VCAM-1 and ICAM-1 in endothelial cells. However, the surface presentation of these adhesion receptors is modulated in an ephrinA1-dependent manner. In conclusion, these data demonstrate that ephrinA1 plays an important role in the TNF-α-mediated adhesion of monocytes to endothelial cells, which might be of great importance in the context of atherosclerosis.