Receptor-mediated adhesion involves both mechanical and chemical signals occurring at the cell-substrate interface. Using a relatively new technique called traction force microscopy, the magnitude, direction, and spatial location of mechanical forces exerted by endothelial cells on a RGD-peptide-derivatized hydrogel substrate were measured. We constructed a surface with a controlled density of cell adhesion nonapeptide containing RGD, which can ligate endothelial cell integrin receptors and induce cell spreading. Increasing the concentration of the RGD peptide increases cell spreading on an otherwise nonadhesive surface of polyacrylamide, and cell area is a monotonically increasing function of peptide concentration. Correlating the force exerted by the cell to the cell area reveals that force is a linear, increasing function of cell area, with a mean increase in cell force of 10(4) dyn/cm(2) cell area. Additionally, we have found that tractions exerted by endothelial cells are concentrated at the ends of pseudopodia and are almost negligible under the nucleus. These results indicate that endothelial cells may have an internal structure that contacts and pulls on the substrate at concentrated locations within the tips of cell extensions and that the strength in adhesion increases with cell spreading.