Eph-related receptors and their ephrin ligands are highly conserved protein families which play important roles in targeting axons and migrating cells. In this study we have examined the functional roles of two major autophosphorylation sites, Tyr-615 and Tyr-838, in the EphA8 receptor. Two-dimensional phosphopeptide mapping analysis demonstrated that Tyr-615 and Tyr-838 constitute major autophosphorylation sites in EphA8. Tyr-615 was phosphorylated to the highest stoichiometry, suggesting that phosphorylation at this site may have a physiologically important role. Upon conservative mutation of Tyr-838 located in the tyrosine kinase domain, the catalytic activity of EphA8 was strikingly reduced both in vitro and in vivo, whereas a mutation at Tyr-615 in the juxtamembrane domain did not impair the tyrosine kinase activity. In vitro binding experiments revealed that phosphorylation at Tyr-615 in EphA8 mediates the preferential binding to Fyn-SH2 domain rather than Src and Ras GTPase-activating protein (Ras GAP)-SH2 domains. Additionally, a high level of EphA8 was detected in Fyn immunoprecipitates in intact cells, indicating that EphA8 and Fyn can physically associate in vivo. In contrast, the association of full-length Fyn to EphA8 containing mutation at either Tyr-615 or Tyr-838 was greatly reduced. These data indicate that phosphorylation of Tyr-615 is critical for determining the association with Fyn whereas the integrity of Tyr-838 phosphorylation is required for efficient phosphorylation at Tyr-615 as well as other major sites. Finally, it was observed that cell attachment responses are attenuated by overexpression of wild type EphA8 receptor but to much less extent by EphA8 mutants lacking phosphorylation at either Tyr-615 or Tyr-838. Furthermore, transient expression of kinase-inactive Fyn in EphA8-overexpressing cells blocked cell attachment responses attenuated by the EphA8 signaling. We therefore propose that Fyn kinase is one of the major downstream targets for the EphA8 signaling pathway leading to a modification of cell adhesion, and that autophosphorylation at Tyr-838 is critical for positively regulating the EphA8 signaling event.