Protein palmitoylation plays a critical role in sorting and targeting of several proteins to pre-and postsynaptic sites. In this study, we have analyzed the role of palmitoylation in trafficking of synaptotagmin I and its modulation by synaptic activity. We found that palmitoylation of N-terminal cysteines contributed to sorting of synaptotagmin I to an intracellular vesicular compartment at the presynaptic terminal. Presynaptic targeting is a unique feature of N-terminal sequences of synaptotagmin I because the palmitoylated N terminus of synaptotagmin VII failed to localize to presynaptic sites. We also found that palmitate was stably associated with both synaptotagmin I and SNAP-25 and that rapid neuronal depolarization did not affect palmitate turnover on these proteins. However, long-term treatment with drugs that either block synaptic activity or disrupt SNARE complex assembly modulated palmitoylation and accumulation of synaptotagmin I at presynaptic sites. We conclude that palmitoylation is involved in trafficking of specific elements involved in transmitter release and that distinct mechanisms regulate addition and removal of palmitate on select neuronal proteins.Synaptic transmission requires appropriate protein targeting and assembly of pre-and postsynaptic elements. Protein sorting to distinct domains in polarized cells appears to begin in the Golgi/trans-Golgi network, where proteins can segregate and exit in separate transport vesicles (1). One mechanism that regulates protein trafficking is palmitoylation, a post-translational modification involving the addition of palmitate, a 16-carbon fatty acid, via a labile thioester linkage (2-5). In neuronal cells, palmitoylation is critical for sorting of several synaptic proteins (6). These include the postsynaptic density protein PSD-95, the AMPA 1 receptor-binding protein, and the presynaptic proteins GAP-43 (growth-associated protein of 43 kDa) and GAD-65 (7-11). Palmitoylation of the AMPA receptor-binding protein and PSD-95 is essential for clustering at the PSD (8 -10), whereas palmitoylation of GAD-65 is important for presynaptic targeting (11,12).Acylation of several other axonal proteins as well as proteins associated with neurotransmitter release machinery has been recently reported (5, 6, 13). These include members of the synaptotagmin family that regulate synaptic vesicle trafficking and neurotransmitter release (14, 15). The synaptotagmin family includes 13 members characterized by a unique N-terminal region followed by a transmembrane domain, a cluster of cysteines (the putative palmitoylation site), a variable domain, and two C-terminal C2 domains (15-18). Synaptotagmin I, the most characterized member of the family, is proposed to act as a Ca 2ϩ sensor for regulated exocytosis (19). Other abundant members of the family include synaptotagmins III and VII (15). Interestingly, synaptotagmin I is localized to synaptic vesicles, whereas synaptotagmin VII is localized on the plasma membrane opposite synaptic vesicle docking sites (15). Despite th...