Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H + )ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K + diffusion gradient. Consequently, the H + -pumping activity of the (H + )ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO 3 - to electrically short-circuit the (H + )ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K m ~5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO 3 - >I - ~ClO 3 - ~Br - >Cl - ~NO 2 - >Iminodiacetate~SO 4 2 - ). Passive NO 3 - transport was also determined, independently of the (H + )ATPase, from the NO 3 - -dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K m for NO 3 - and should be suitable to continue the functional and biochemical characterization of the NO 3 - transport system.