Abstract: The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three dimensional model of the intact V-ATPase was calculated from two dimensional projections of the complex at a resolution of 25 A. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo hexagonal arrangement in the V1, the N-terminus of subunit G in the V1-V0 interface and the C-terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available X-ray crystal structures into the electron microscopy derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.