The vacuolar H(+)-ATPase (V-ATPase) is a rotary motor enzyme that acidifies intracellular organelles and the extracellular milieu in some tissues. Besides its canonical proton-pumping function, V-ATPase's membrane sector, Vo, has been implicated in non-canonical functions including membrane fusion and neurotransmitter release. Here, we report purification and biophysical characterization of yeast V-ATPase c subunit ring (c-ring) using electron microscopy and single-molecule electrophysiology. We find that yeast c-ring forms dimers mediated by the c subunits' cytoplasmic loops. Electrophysiology measurements of the c-ring reconstituted into a planar lipid bilayer revealed a large unitary conductance of ~8.3 nS. Thus, the data support a role of V-ATPase c-ring in membrane fusion and neuronal communication.