Neurotransmission requires precise assembly of receptors and associated signal transduction machinery at synaptic sites. At excitatory synapses, glutamate receptors and downstream enzymes are clustered in functional units at the PSD, and this allows for rapid and specific synaptic responses. In addition to facilitating efficient signaling, the PSD retains a remarkable capacity for structural reorganization, which is essential for aspects of synaptic plasticity. Whereas biochemical studies have helped define the composition of the PSD, mechanisms that mediate postsynaptic protein targeting remain largely unknown. To address this important question, our laboratory has initiated a program of cell biological studies to analyze synaptic targeting of the postsynaptic density protein, PSD-95. PSD-95 is a membrane-associated guanylate kinase that employs PDZ protein motifs to cluster ion channels at synaptic sites. We find that postsynaptic targeting relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. The requirement for palmitoylation, the post-translational attachment of two 16 carbon saturated fatty acids, is particularly striking as palmitoylation cannot be replaced with alternative membrane association motifs. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting. Taking advantage of these preliminary data and our ability to monitor synaptic protein trafficking in cultured neurons, we propose now to define the underlying molecular mechanisms by which palmitoylation, PDZ domains and the C-terminal motif determine synaptic targeting of PSD-95. This work will provide insight into fundamental mechanisms that underlie synaptic protein assembly. Also, because PSD-95 organizes signal transduction cascades downstream of the N-methyl-D-aspartate (NMDA) type glutamate receptor, this proposal has relevance for synaptic plasticity and neurodegeneration mediated by the NMDA receptor.