Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08 to 1.15 grams per cubic centimeter with maximum activity at 1.10 to 1.12 grams per cubic centimeter. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing cytochrome c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing beta-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-cytochrome c reductase and nucleoside diphosphatase activities. It is suggested that the vesicles are of tonoplast origin.