Cleared maize (Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP- and pyrophosphate (PPi)-dependent proton-pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PPi-dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H+-PPase was Mg2+-dependent and the Km for PPi (in the presence of 3 mM MgSO4) was 28 μM. The pump was electrogenic, K+-dependent and a permeant anion was necessary to dissipate the membrane potential (NO3−= I− >Br− > Cl−). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H+-PPase was insensitive to vanadate, oligomycin and molybdate. -Diethylstilbestrol (DES) and N,N′-dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μM.