The pH-dependent fluorescence quenching of acridine orange was used to study the Na(+)- and K(+)-dependent H(+) fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na(+)-dependent H(+) flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na(+) concentration and was competitively inhibited by amiloride (K(i) = 0.1 millimolar). The potential-dependent component of H(+) flux showed an approximately linear dependence on Na(+) concentration. In contrast, the K(+)-dependent H(+) flux apparently consisted of a single component which showed an approximately linear dependence on K(+) concentration, and was insensitive to amiloride. Based on the Na(+)- and K(+)-dependent H(+) fluxes, the passive permeability of the vesicle preparation to Na(+) was about half of that to K(+).The apparent K(m) for Na(+) of the electroneutral Na(+)/H(+) exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na(+)/H(+) antiport which can account for the estimated in vivo accumulation ratio for Na(+) into the vacuole.