The biophysical properties of the interaction between fibronectin and its membrane receptor were inferred from adhesion tests on living cells. Individual fibroblasts were maintained on fibronectin-coated glass for short time periods (1-16 s) using optical tweezers. After contact, the trap was removed quickly, leading to either adhesion or detachment of the fibroblast. Through a stochastic analysis of bond kinetics, we derived equations of adhesion probability versus time, which fit the experimental data well and were used to compute association and dissociation rates (k+ = 0.3-1.4 s(-1) and koff = 0.05-0.25 s(-1), respectively). The bond distribution is binomial, with an average bond number < or = 10 at these time scales. Increasing the fibronectin density (100-3000 molecules/microm2) raised k+ in a diffusion-dependent manner, leaving koff relatively unchanged. Increasing the temperature (23-37 degrees C) raised both k+ and koff, allowing calculation of the activation energy of the chemical reaction (around 20 kBT). Increasing the compressive force on the cell during contact (up to 60 pN) raised k+ in a logarithmic manner, probably through an increase in the contact area, whereas koff was unaffected. Finally, by varying the pulling force to detach the cell, we could distinguish between two adhesive regimes, one corresponding to one bond, the other to at least two bonds. This transition occurred at a force around 20 pN, interpreted as the strength of a single bond.