Activation of tyrosine kinases during integrin-mediated cell-matrix adhesion is involved both in the regulation of focal contact assembly and in the initiation of signaling processes at the cell-matrix adhesive interface. In order to determine the role of pp60(c-src) and related kinases in these processes, we have compared the dynamic reorganization of phosphotyrosine, vinculin, focal adhesion kinase and tensin in cells with altered expression of Src-family kinases. Both null cells for pp60(c-src) and triple knockout cells for pp60(c-src), pp59(fyn), and pp62(c-yes) exhibited decreased phosphotyrosine levels in focal contacts when compared with wild-type cells. pp60(c-src)-null cells also exhibited faster assembly of cell-matrix adhesions and a more exuberant recruitment of FAK to these sites. Tensin, which normally segregates into fibrillar adhesions was localized in large focal contacts in the two mutant cell lines, suggesting involvement of pp60(c-src) in the segregation of focal contacts and fibrillar adhesions. Moreover, treatment of wild-type cells with tyrphostin AG1007, which inhibits both pp60(c-src) and FAK activity, induced accumulation of tensin in peripheral focal adhesions. These findings demonstrate that Src family kinases, and pp60(c-src) in particular, have a central role in regulating protein dynamics at cell-matrix interfaces, both during early stages of interaction and in mature focal contacts.