Crk-associated substrate (CAS) is a tyrosine kinase substrate implicated in integrin control of cell behavior. Phosphorylation, by Src family kinases, of multiple tyrosine residues in the CAS substrate domain (SD) is a major integrin signaling event that promotes cell motility. In this study, novel phosphospecific antibodies directed against CAS SD phosphotyrosine sites ("pCAS" antibodies) were characterized and employed to investigate the cellular regulation and localization of CAS SD tyrosine phosphorylation. An analysis of CAS and focal adhesion kinase (FAK) variants expressed in CAS- and FAK-deficient cell lines, respectively, indicated that CAS SD tyrosine phosphorylation is substantially achieved by Src family kinases brought into association with CAS through two distinct mechanisms: direct binding to the CAS Src-binding domain and indirect association through a FAK bridge. Cell immunostaining with pCAS antibodies revealed that CAS SD tyrosine phosphorylation occurs exclusively at sites of integrin adhesion including both nascent focal complexes formed at the edges of extending lamellipodia as well as mature focal adhesions underlying the cell body. These findings further document a role for FAK as an important upstream regulator of CAS SD tyrosine phosphorylation and implicate CAS-mediated signaling events in promoting membrane protrusion/lamellipodium extension during cell motility.