Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP-dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.