The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [14C]triphenylmethyl-phosphonium ion ([14C]TPMP +) and [3H]tetraphenyl-phosphonium ion ([3H]TPP ยง by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [~4C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of transmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N'-dicyclohexylcarbodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3tert-butyl-2"-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H ยง pump is catalyzed by the tonoplast ATPase of higher plants.