The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin alphavbeta3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCgamma in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MPhis) treated with tumor necrosis factor-alpha (TNFalpha) or interleukin-1beta (IL1beta). RA and OA STs showed a greater percentage of pFAK on lining cells and MPhis compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFalpha or IL1beta stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MPhis. pPyk2 was expressed on RA ST fibrobasts but not in MPhis at baseline, however it was upregulated upon TNFalpha or IL1beta activation in both cell types. pSrc was expressed in RA ST fibroblasts and MPhis at baseline and was further increased by TNFalpha or IL1beta stimulation. pPaxillin and pPLCgamma were upregulated in RA versus OA and ND lining cells and sublining MPhis. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.