The Eph receptors are the largest known family of receptor protein tyrosine kinases, which play important roles with their ligands called ephrin in the neural development, angiogenesis, and vascular network assembly. It was previously shown that ephrin-A2, -A3 and -A5 bind to, and activate the EphA8 receptor tyrosine kinase, respectively. In this study, we have examined if there are other additional ephrin ligands interacting with the EphA8 receptor tyrosine kinase expressed in NIH3T3 fibroblasts. For this purpose, we have constructed chimeric ephrin-A1, -A4, -B1, -B2 or -B3 ligands consisting of the Fc portion of human IgG fused to their carboxyl-terminus. Both ephrin-A1 and ephrin-A4 chimeric ligands efficiently bound to the EphA8 receptor expressed in NIH3T3 fibroblasts, whereas the transmembrane ligands including ephrin-B1, -B2 and -B3 did not. Additionally we have demonstrated that both the EphA8-TrkB chimeric receptor and the EphA8 receptor expressed in NIH3T3 fibroblasts are efficiently tyrosine-phosphorylated upon stimulating with epthin-A1 or -A4 but none of transmembrane ephrin-B proteins. These results strongly indicate that the EphA8 receptor functions exclusively as an glycosyl phosphatidylinositol (GPI)-linked ephrin ligand-dependent receptor protein tyrosine kinase.