We report for the first time an atomic force microscopy (AFM) investigation of the dependence of unbinding force on temperature at controlled loading rate. AFM force measurements of biotin-avidin interactions were acquired at various temperatures ranging from 13 to 37 degreesC using force loading rates that are slow enough to ignore dissipative friction and to assume thermal equilibrium. With our established Poisson statistical analysis method, the unbinding force at a fixed loading rate between an individual biotin-avidin pair was extracted and was found to decrease by similar to5-fold with increasing temperature over this range. On the basis of a thermodynamic model similar to that of Bell, but developed here with additional insights, the bond stiffness, effective unbinding length, and the critical unbinding energy per biotin-avidin complex were estimated. This work takes quantitative AFM studies to the next level by allowing energies to be determined from force measurements.