To characterize the dynamics of cell-substrate adhesive rupture, we used a novel micromanipulation technique, in which individual fibroblasts seized on a rigid microplate were placed into contact with a fibronectin-coated flexible microplate, then pulled away. The fibronectin density (0-3000 molecules/microm2) and the pulling rate (1-10 microm/s) were controlled. The extent of the contact zone decreased to zero at a time threshold corresponding to adhesive rupture. The uniaxial force at the interface, computed from the deflection of the microplate, increased linearly with time and reached a maximum before dropping to zero. A deterministic model, focusing on the mean number of bonds between fibronectin and its membrane receptor on the cell surface, shows rapid rupture when the force reaches a critical value, in agreement with experimental observations. Increasing the ligand density and the rate of load raises the maximal force (30 200 nN), in reasonably good agreement with the model predictions. Minimization of error between experimental and simulated forces allowed identification of two physicochemical properties of the bond, i.e. its association rate constant (k(2D)on = 3 x 10(-4) microm2/s) and structural length (d = 3 nm). These results may help understand better fibroblast locomotion and interaction with the extracellular matrix.