This study investigates how substrate stiffness affects the morphology, cytoskeletal structure, and adhesion of fibroblasts, endothelial cells, and neutrophils. Cells were cultured on substrates with stiffness ranging from 2 to 55,000 Pa, laminated with fibronectin or collagen. Fibroblasts and endothelial cells showed a sudden change in spread area around 3,000 Pa stiffness, with no actin stress fibers on soft surfaces and their appearance coinciding with the stiffness range where they spread. α5 integrin expression also increased in this range, but exogenous α5 integrin expression was not sufficient to cause spreading on soft surfaces. Neutrophils, however, showed no dependence of shape or spreading ability on substrate stiffness. The differences in morphology and cytoskeletal structure on soft versus hard substrates were eliminated when cells made cell-cell contact. These findings support the hypothesis that mechanical factors influence different cell types in distinct ways, triggering changes similar to those caused by soluble ligands. The study also highlights the importance of substrate stiffness in cell growth, survival, and responses to mechanical stimuli, with variations in protein tyrosine phosphorylation and cytoskeletal structure correlating with stiffness. The results suggest that cell behavior is significantly influenced by substrate stiffness, with different cell types responding in distinct ways. The study used polyacrylamide gels with controlled stiffness and adhesion ligands to examine these effects, showing that cell morphology and cytoskeletal structure vary with substrate stiffness. The findings have implications for understanding cell behavior in vivo and the role of mechanical signals in cell function.This study investigates how substrate stiffness affects the morphology, cytoskeletal structure, and adhesion of fibroblasts, endothelial cells, and neutrophils. Cells were cultured on substrates with stiffness ranging from 2 to 55,000 Pa, laminated with fibronectin or collagen. Fibroblasts and endothelial cells showed a sudden change in spread area around 3,000 Pa stiffness, with no actin stress fibers on soft surfaces and their appearance coinciding with the stiffness range where they spread. α5 integrin expression also increased in this range, but exogenous α5 integrin expression was not sufficient to cause spreading on soft surfaces. Neutrophils, however, showed no dependence of shape or spreading ability on substrate stiffness. The differences in morphology and cytoskeletal structure on soft versus hard substrates were eliminated when cells made cell-cell contact. These findings support the hypothesis that mechanical factors influence different cell types in distinct ways, triggering changes similar to those caused by soluble ligands. The study also highlights the importance of substrate stiffness in cell growth, survival, and responses to mechanical stimuli, with variations in protein tyrosine phosphorylation and cytoskeletal structure correlating with stiffness. The results suggest that cell behavior is significantly influenced by substrate stiffness, with different cell types responding in distinct ways. The study used polyacrylamide gels with controlled stiffness and adhesion ligands to examine these effects, showing that cell morphology and cytoskeletal structure vary with substrate stiffness. The findings have implications for understanding cell behavior in vivo and the role of mechanical signals in cell function.