We have investigated how extracellular matrix (ECM) alters the mechanical properties of the cytoskeleton (CSK). Mechanical stresses were applied to integrin receptors on the apical surfaces of adherent endothelial cells using RGD-coated ferromagnetic microbeads (5.5-microns diameter) in conjunction with a magnetic twisting device. Increasing the number of basal cell-ECM contacts by raising the fibronectin (FN) coating density from 10 to 500 ng/cm2 promoted cell spreading by fivefold and increased CSK stiffness, apparent viscosity, and permanent deformation all by more than twofold, as measured in response to maximal stress (40 dyne/cm2). When the applied stress was increased from 7 to 40 dyne/cm2, the stiffness and apparent viscosity of the CSK increased in parallel, although cell shape, ECM contacts, nor permanent deformation was altered. Application of the same stresses over a lower number ECM contacts using smaller beads (1.4-microns diameter) resulted in decreased CSK stiffness and apparent viscosity, confirming that this technique probes into the depth of the CSK and not just the cortical membrane. When magnetic measurements were carried out using cells whose membranes were disrupted and ATP stores depleted using saponin, CSK stiffness and apparent viscosity were found to rise by approximately 20%, whereas permanent deformation decreased by more than half. Addition of ATP (250 microM) under conditions that promote CSK tension generation in membrane-permeabilized cells resulted in decreases in CSK stiffness and apparent viscosity that could be detected within 2 min after ATP addition, before any measurable change in cell size. Permanent deformation only decreased after 20 min, once the CSK lattice had physically contracted. Importantly, regardless of cell shape or membrane continuity, CSK stiffness increased in direct proportion to the applied stress, as predicted by tensegrity (tensional integrity) cell models. These results suggest that the effects of ECM on CSK mechanics are not due to changes in osmotic or hydrostatic pressures. Rather, ECM alters CSKstiffness and apparent viscosity by binding integrins, promoting formation of molecular links with the CSK, transmitting mechanical stresses across these linkages, and inducing structural rearrangements within a continuous, tensionally integrated CSK lattice. In contrast, permanent deformation in the CSK appears to be more tightly coupled to cell extension and depends on both passive plasticity and dynamic remodeling events.