Supramolecular 15 nm cup‐shaped lipoprotein structures called porosomes present at the presynaptic membrane, mediate neurotransmitter release via the transient fusion of synaptic vesicles at its base via SNAREs. Major lipids associated with the isolated neuronal porosome complex have been determined to be unique in composition. Since lipids are known to greatly influence various membrane protein association and functions including membrane fusion, the composition of lipids in synaptic vesicle and the synaptosome membrane, was hypothesized to be unique and tightly regulated. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicles, was determined using mass spectrometry. Results from the study demonstrate a unique lipid composition of the synaptic vesicle compared to the synaptosome membrane, reflecting on the tight regulation of nerve cells in maintaining the lipid composition of both organelles.Support or Funding InformationWork presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ); the WSU Interdisciplinary Biomedical Systems Fellowship and the Thomas C. Rumble University Graduate Fellowship (KTL); and the WSU Interdisciplinary Biomedical Systems Fellowship (ARN).