The fibronectin binding integrins α5β1 and α4β1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For α5β1, β1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for α5β1-stimulated cell motility and that exogenous expression of human α4 in FAK-null fibroblasts forms a functional α4β1 receptor that promotes robust cell motility equal to the α5β1 stimulation of wild-type and FAK-reconstituted fibroblasts. α4β1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the α4 cytoplasmic domain, independent of direct paxillin binding to α4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. α4 cytoplasmic domain-initiated signaling led to a ∼4-fold activation of c-Src which did not require paxillin binding to α4. Notably, α4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase α over-expression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. α4β1-stimulated cell motility of triple-null Src-/-, c-Yes-/-, and Fyn -/- fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for α5β1-stimulated FAK activation, our results support the existence of a novel α4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway. Copyright © 2005, American Society for Microbiology. All Rights Reserved.