The Eph family of receptor tyrosine kinases and their cognate ligands, the Ephrins, form a coordinated program of cell contact-mediated migration control, polarity establishment, and tissue architecture development. Specifically, the ligand Ephrin-A1 has been shown to regulate cell morphology and motility through the activation of EphA receptors, which signal to the PI3K pathway to induce cell retraction. MEFs with PI3K subunit p85b knockout (p85b À/À ) exhibited markedly reduced cell retraction following Ephrin-A1 stimulation. Ephrin-A1 also serves as an inhibitory substrate for cell spreading and migration. Moreover, Ephrin-A1 treatment results in the dephosphorylation of paxillin and induces the reorganization of phosphopaxillin-containing focal adhesions. The Ephrin-A1 regulated paxillin dephosphorylation is phosphatase dependent, but p85b independent. The present study serves to demonstrate a novel molecular signaling pathways that regulate Ephrin-A1-regulated cell retraction and interaction to the substrate.