Single molecule experiments have opened promising new avenues of investigations in biology, but the quantitative interpretation of results remains challenging. In particular, there is a need for a comparison of such experiments with theoretical methods. We experimentally determine the activation free energy for single molecule interactions between two synaptic proteins syntaxin 1A and synaptobrevin 2, using an atomic force microscope and the Jarzynski equality of nonequilibrium thermodynamics. The value obtained is shown to be reasonably consistent with that from single molecule reaction rate theory. The temperature dependence of the spontaneous dissociation lifetime along with different pulling speeds is used to confirm the approach to the adiabatic limit. This comparison of the Jarzynski equality for intermolecular interactions extends the procedure for calculation of activation energies in nonequilibrium processes. © 2008 by the Biophysical Society.