Large-scale proteomic analyses of heterogeneous CNS preparations, such as isolated CNS synaptic vesicles, have provided a wealth of information related to synapse specific proteins, and synaptic signaling pathways. However, these studies under-represent, or lack entirely, many vesicle and neurotransmitter signaling pathways. In particular the neuromodulatory adrenergic and cholinergic pathways are often under-represented. In order to better understand the molecules and signaling pathways involved at cholinergic synapses, we have focused on the classic preparation of the electric lobe and electric organ of Torpedo californica. The abundant macromolecular material available from this preparation has contributed greatly to our understanding of the synthesis, storage, and release of the neurotransmitter acetylcholine and its postsynaptic activation of the nicotinic acetylcholine receptor. Many lines of evidence support the idea that in addition to acetylcholine, additional neurotransmitters and/or neuromodulators are also released from cholinergic synapses. Utilizing a single-vesicle imaging approach, we have found that individual synaptic vesicles from the electric ray possess neurotransmitter transporters for glutamate, ATP, and acetylcholine. In addition to those transporters, cholinergic synaptic vesicles from the electric ray also possess an orphaned transporter from the bile acid transport family, SLC10A4. We are now expanding our research to identify the molecules transported by SLC10A4 using multiple biochemical approaches and a mouse knockout model.