Cellular interactions with the extracellular matrix proteins play important roles in a variety of biological processes. Recent studies suggest that integrin-mediated cell-matrix interaction can transduce biochemical signals across the plasma membrane to regulate cellular functions such as proliferation, differentiation and migration. These studies have implicated a critical role of focal adhesion kinase (FAK) in integrin-mediated signal transduction pathways. We report here that overexpression of FAK in CHO cells increased their migration on fibronectin. A mutation of the major autophosphorylation site Y397 in FAK abolished its ability to stimulate cell migration, while phosphorylation of Y397 in a kinase-defective FAK by endogenous FAK led to increased migration. We also find that the wild-type and the kinase-defective FAK were associated with Src and Fyn in CHO cells whereas the F397 mutant was not. These results directly demonstrate a functional role for FAK in integrin signaling leading to cell migration. They also provide evidence for the functional significance of FAK/Src complex formation in vivo.