We present evidence strongly suggesting that a proton gradient (acid inside) is used to drive an electroneutral, substratespecific, K+/H antiport in both tonoplast and plasma membraneenriched vesicles obtained from oilseed rape (Brassica napus) hypocotyls. Proton fluxes into and out of the vesicles were monitored both by following the quenching and restoration of quinacrine fluorescence (indicating a transmembrane pH gradient) and of oxonol V fluorescence (indicating membrane potential.) Supply of K+ (with Cl-or SCN-) after a pH gradient had been established across the vesicle membrane by provision of ATP to the H+-ATPase dissipated the transmembrane pH gradient but did not depolarize the positive membrane potential. Evidence that the K+/H+ exchange thus indicated could not be accounted for by mere electric coupling included the findings that, first, no positive potential was generated when KSCN or KCI was supplied, even in the absence of 100 millimolar Cl-and, second, efflux of K+ from K+-loaded vesicles drives intravesicular accumulation of H+ against the electrochemical potential gradient. Neither was the exchange due to competition between K+ and quinacrine for membrane sites, nor to inhibition of the H+-ATPase. Thus, it is likely that it was effected by a membrane component. The exchanger utilized primarily K+ (at micromolar concentrations); Na+/ H+ antiport was detected only at concentrations two orders of magnitude higher. Rb+, Li+, or Cs+ were ineffective. Dependence of tonoplast K+/H+ antiport on K+ concentration was complex, showing saturation at 10 millimolar K+ and inhibition by concentrations higher than 25 millimolar. Antiport activity was associated both with tonoplast-enriched membrane vesicles (where the proton pump was inhibited by more than 80% by 50 millimolar N03-and showed no sensitivity to vanadate or oligomycin) and with plasma membrane-enriched fractions prepared by phase separation followed by separation on a sucrose gradient (where the proton pump was vanadate and diethylstilbestrol-sensitive but showed no sensitivity to N03-or oligomycin). The possible physiological role of such a K+/H exchange mechanism is discussed.According to the chemiosmotic hypothesis, currently widely accepted, a primary membrane-sited H+-translocating ATPase generates a transmembrane electrochemical potential gradient for protons; the return flux of the protons drives the transmembrane flux of other solutes via secondary symport or antiport mechanisms. Although good evidance at isolated membrane level has been obtained for the presence and function of H+-ATPases in higher plant cell membranes (25), evidence at this level for secondary systems transporting ions has been more difficult to attain, with the exception of the tonoplast-associated Ca2+/H' antiporter (4, 20, 23). With regard to monovalent cation flux, there are good indications for the existence of Na+/H+ antiporters in the case of several halophytes or salt-tolerant species (3,6,11,12,13). The presence of these mechanisms has been related ...