V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V(0)), and a soluble catalytic (V(1)) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V(0) and V(1) were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V(0) changed with respect to V(1), and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly.