This study investigates the effects of substrate stiffness on the morphology, cytoskeletal structure, and adhesion of fibroblasts, endothelial cells, and neutrophils. The cells were cultured on polyacrylamide gels with stiffness ranging from 2 to 55,000 Pa, laminated with fibronectin or collagen. Fibroblasts and endothelial cells showed an abrupt change in spread area at a stiffness of around 3,000 Pa, with the appearance of stress fibers coinciding with this stiffness range. Neutrophils, however, did not show a dependence on substrate stiffness up to 2 Pa. The shape and cytoskeletal differences observed in single cells on soft versus hard substrates were eliminated when fibroblasts or endothelial cells made cell-cell contacts. These findings suggest that mechanical factors impact different cell types differently and can trigger specific changes similar to those stimulated by soluble ligands. The study also highlights the importance of the nature of the adhesion ligand and the role of integrin expression in these responses.This study investigates the effects of substrate stiffness on the morphology, cytoskeletal structure, and adhesion of fibroblasts, endothelial cells, and neutrophils. The cells were cultured on polyacrylamide gels with stiffness ranging from 2 to 55,000 Pa, laminated with fibronectin or collagen. Fibroblasts and endothelial cells showed an abrupt change in spread area at a stiffness of around 3,000 Pa, with the appearance of stress fibers coinciding with this stiffness range. Neutrophils, however, did not show a dependence on substrate stiffness up to 2 Pa. The shape and cytoskeletal differences observed in single cells on soft versus hard substrates were eliminated when fibroblasts or endothelial cells made cell-cell contacts. These findings suggest that mechanical factors impact different cell types differently and can trigger specific changes similar to those stimulated by soluble ligands. The study also highlights the importance of the nature of the adhesion ligand and the role of integrin expression in these responses.