The integrins are heterodimeric transmembrane receptors that are important for a number of biological processes. Interaction of these receptors with extracellular matrix (ECM) proteins results in the activation of intracellular signaling pathways. Focal Adhesion Kinase(FAK) is a protein tyrosine kinase that appears to be closely linked with integrin-mediated signaling. It can associate with the Src family members c-Src and c-Fyn, and the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI-3K). The p70/p85 ribosomal S6 kinase (S6K) is a mitogen-activated Ser/Thr kinase that phosphorylates the 40 S ribosomal subunit protein S6 and plays a critical role in cell proliferation. We report that S6K can be activated in an integrin-dependent manner as determined by cell spreading on an extracellular matrix. Results obtained by treating cells with inhibitors of PI-3K and FRAP (FKBP 12/Rapamycin associated protein) suggest that these proteins are required for the integrin-mediated activation of S6K. Inhibitor studies also indicate that tyrosine kinase activity is required for S6K activation. Since FAK can interact with Src family kinases, we examined the role of this bipartite kinase complex in the integrin-directed activation of S6K. Overexpression of FAK Related Non Kinase (FRNK) in chick embryo fibroblast (CEF) cells results in a 4-5 fold reduction in the phosphotyrosine content of FAK, thus functioning as a dominant negative inhibitor of FAK tyrosine phosphorylation. Under these conditions we detected a 50% reduction in S6K activation. To determine the role of c-Src, we overexpressed c-Src and the Src-RF dominant negative mutant in CEF cells. We detected a delay in the maximal S6K activation in cells overexpressing Src-RF to 60 minutes after replating onto fibronectin, and the magnitude of activation at 30 min was approximately 60% of the level detected in mock transfected cells. Conversely, c-Src overexpression resulted in a fibronectin-dependent potentiation of S6K activity. These results suggest that both FAK and c-Src can contribute to S6K activation and mediate crosstalk between integrin- and mitogen-activated signaling. The integrin-mediated activation of S6K may be important in providing a survival signal for the cell.