Cholesterol has been proposed to play a critical role in regulating neurotransmitter release and synaptic plasticity. The neuronal porosome/fusion pore, the secretory machinery at the nerve terminal, is a 12–17 nm cup-shaped lipoprotein structure composed of cholesterol and a number of proteins, among them calcium channels, and the t-SNARE proteins Syntaxin-1 and SNAP-25. During neurotransmission, synaptic vesicles dock and fuse at the porosome via interaction of their v-SNARE protein with t-SNAREs at the porosome base. Membrane-associated neuronal t-SNAREs interact in a circular array with liposome-associated neuronal v-SNARE to form the t-/v-SNARE ring complex. The SNARE complex along with calcium is required for the establishment of continuity between opposing bilayers. Here we show that although cholesterol is an integral component of the neuronal porosome and is required for maintaining its physical integrity and function, it has no influence on the conformation of the SNARE ring complex.