Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg(2+)-ATPase activity of the sealed vesicles was stimulated by Cl(-) and NH(4) (+) and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO(3) (-), was insensitive to K(+), and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg(2+), was stimulated by Cl(-), inhibited by NO(3) (-), was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg(2+) >/= Mn(2+) > Co(2+)) and were inhibited by high concentrations of Ca(2+). The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F(1)F(0)-ATPase and from those of the K(+)-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.