Mechanical properties of the extracellular matrix (ECM) are proposed to influence cell behavior and biological activity. The influence of the mechanical environment on fibronectin fibril assembly was evaluated. Fibroblasts were cultured in hydrated collagen gels with two distinctly different mechanical properties. Cells cultured within a stabilized collagen gel generate stress that is transmitted throughout the matrix (stressed gel). In contrast, cells that are cultured within a collagen gel that is floating freely in media do not generate stress (relaxed gel). Fibroblasts in the stressed collagen gel develop large bundles of actin microfilaments and associated fibronectin fibrils, while fibroblasts within relaxed gels do not form stress fibers or assemble fibronectin into fibrils. In addition, we have evaluated the mechanism of fibronectin fibril assembly employed by fibroblasts cultured within a stressed three-dimensional collagen matrix and the role of fibronectin fibrils in transmission of cell-generated forces to the surrounding matrix. Fibronectin fragments (70-kDa amino terminal fragment, 110-kDa cell-adhesive fragment, and GRGDS peptide) and a monoclonal anti-body blocked fibronectin fibril assembly in stressed three-dimensional collagen gels. These results suggest that the features of fibronectin required for fibronectin fibril assembly by cells in collagen gels is similar to those required by cells cultured on a planar substratum. Although fibronectin fibril assembly was blocked by these inhibiting fragments and antibody, the cells displayed prominent actin bundles and developed isometric tension, indicating that stress fiber formation and contractile force transmission is not dependent on the presence of fibronectin fibrils.