We have previously demonstrated that binding of ephrin-A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin-A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin-A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin-A1-induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion-like kinase proline-rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin-A1-induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho-associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.