AbstractTonoplast membranes were prepared from tissue homogenates and from vacuoles isolated from beetroot storage tissue (Beta vulgarisL., ssp.conditiva) for transmission electron microscopic analysis of the structure of the beetroot vacuolar ATPase using the negative staining technique. By comparison of the specific inhibitor sensitivities of the ATPase activity, i.e. ATP hydrolysis and H+‐pumping, the purity of the tonoplast preparations with respect to contamination with mitochondrial inner membranes was assessed to avoid confusion with mitochondrial F1F0‐ATPase. Membranes prepared in Hepes/Tris or BTP/Mes‐containing media rarely showed typical head‐and‐stalk structures although characteristic nitrate‐ and bafilomycin A1‐sensitive ATP‐hydrolysis and H+‐pumping could be measured. However, typical head‐and‐stalk structures were observed regularly when these buffers were replaced by K‐phosphate buffer. Under these conditions, the beetroot vacuolar ATPase is characterized by a large head group with a central cleft, a thin stalk, connecting it to the membrane and by basal components projecting from the base of the stalks near the vacuolar membrane and forming a distinct layer of electron‐light particles between the vacuolar membrane and the layer o