Tissue remodeling following injury involves TGF-beta-mediated fibroblast contraction. While these cells are embedded in a fibronectin (FN)-rich matrix, the role of FN-cell interactions in this process is not fully understood. To explore the role of FN matrix presentation, we analyzed the effect of TGF-beta on fibroblasts adhered to FN-coated polyacrylamide gels (PAG). Surprisingly, under these conditions TGF-beta triggered cell rounding/contraction. This was accompanied by increased Rho activation and MLC phosphorylation and was reversed by inhibition of Rho kinase. Although fibroblasts are known to bind to fibronectin's RGD and synergy sites, their relative contribution to cell function is not clear. MLC phosphorylation was reduced and cell contraction was reversed when FN's synergy site was blocked, indicating that contraction requires signals from the synergy site in addition to TGF-beta-mediated Rho activation. Thus, regulating the FN synergy site therapeutically may provide a mechanism for modulating contractile forces during tissue repair.