Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and β-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that β-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to β-neurexin and was not observed with other synaptic cell adhesion molecules such as N- cadherin or SynCAM. NMDA receptors, but not α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this β-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic β-neurexin and that glutamate release also is required for maturation of synapses.