Ionotropic glutamate receptors are tetramers, the isolated ligand binding cores of which assemble as dimers. Previous work on nondesensitizing AMPA receptor mutants, which combined crystallography, ultracentrifugation, and patch-clamp recording, showed that dimer formation by the ligand binding cores is required for activation of ion channel gating by agonists. To define the mechanisms responsible for stabilization of dimer assembly in native AMPA receptors, contacts between the adjacent ligand binding cores were individually targeted by amino acid substitutions, using the GluR2 crystal structure as a guide to design mutants. We show that disruption of a salt bridge, hydrogen bond network, and intermolecular van der Waals contacts between helices D and J in adjacent ligand binding cores greatly accelerates desensitization. Conservation of these contacts in AMPA and kainate receptors indicates that they are important determinants of dimer stability and that the dimer interface is a key structural element in the gating mechanism of these glutamate receptor families.