The synaptic stabilization of AMPA receptors critically depends on the C-terminal interactions of Stargazin (STG) / transmembrane AMPA receptor regulatory protein (TARP) family members with postsynaptic PDZ domain-containing scaffold MAGUK proteins such as PSD-95 (Nicoll et al., 2006, Bats et al., 2007). To investigate the role of these interactions in the dynamics of AMPA receptors synaptic residency we developed biomimetic competing ligands assembled from two PDZ domain-binding motifs of STG. Characterization of the ligands in a cellular fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) system with PSD-95::GFP as donor and STG::mCherry as acceptor revealed an increased affinity for multiple PDZ domains and strong cooperative interaction with PSD-95 (Hill coefficients of 1.378 and 0.9 respectively, for divalent and monovalent ligands). In cultured hippocampal neurons from Wistar E19 rat pups, the divalent PDZ domain ligands competed with endogenous TARP for the intracellular MAGUK. Incubation for five minutes with membrane permeable ligands (5 µM TAT-[STG15]2) acutely increased the lateral diffusion (median diffusion rate 0.059 µm2/s, IQR = 0.021 – 0.16 µm2/s, n=1000 trajectories, vs. 0.018 µm2/s, IQR = 0.049 – 0.11 µm2/s, n=604 trajectories, in control). The effect was transient, peaking at five minutes (mean ± SEM synaptic and extrasynaptic mobile fractions, TAT-[STG15]2: 55.51 ± 3.23%, and 88.21 ± 2.74%, n=7 cells; control: 33.94 ± 5% and 62.96 ± 5.9%, n=8 cells) and disappeared ten minutes after ligand application. This time course was probably due to the entry of mobile AMPARs freed from their PSD-95 anchor in the endocytic pathway, where they would appear as immobile. Indeed, incubation of neurons with TAT-[STG15]2 for 10 min markedly increased the fraction of internalized AMPA receptors (mean ± SEM, 0.84 ± 0.047, n=6 cells vs 0.23 ± 0.06 in controls, n=5 cells). Intracellular delivery of 50 µM [STG15]2 via patch pipettes induced a rundown of AMPA receptor-mediated EPSCs in synaptically coupled neurons. The rundown was progressive and reached equilibrium after 20 minutes at 45 ± 4% of the amplitude during the first minute of recording (n=9). The amplitude of NMDA receptor-mediated EPSCs was not affected. [STG15]2 also induced a progressive rundown of AMPA receptor-mediated miniature EPSCs (mEPSC), that equilibrated after 25 minutes, at 76.7 ± 4% (mean ± SEM, n=6) of the amplitude during the first minute of recording. These results provide evidence for a model in which the TARP-containing AMPA receptors are stabilized at the synapse by engaging in multivalent interactions with the PDZ domain-containing scaffold proteins. In addition, our data suggest the presence of multiple populations of synaptic AMPA receptors that differ in their degree of stabilization.