Interactions between synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) can be readily isolated and studied with the use of force spectroscopy single-molecule measurements. We studied interactions between Sx1A and Sb2 in two different orientations (parallel and antiparallel) using four different terminus configurations of these proteins. Force-loading experiments indicated that protein pairs in any configuration/orientation are zippered. We measured the extension and force for disassembly of these interactions, calculated the spontaneous dissociation lifetimes, and determined their free energies, enthalpies, and entropies. Although the free energies were very similar for all four configurations (∼28 k BT (Eyring model) and ∼20 k BT (Kramers model)), the enthalpy changes of binary Sx1A-Sb2 interactions varied between 24.7 k BT and 33.1 k BT. This variation is consistent with the conformation changes that occur during disassembly of the various protein terminus configurations, as verified by alterations in the extension. The parallel interactions appear to be energetically somewhat advantageous over antiparallel configurations/ orientation, especially when the N-termini of Sx1A-Sb2 are left to interact freely. © 2011 Biophysical Society.