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The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron-SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful "toolkit" for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo.