The plasma membrane ATPases of eukaryotic cells of the vegetable type (fungi, plants, and algae) have been postulated to operate as proton pumps which generate membrane potentials and drive the uptake of nutrients by proton co-transport (Poole, R. J. (1978) Annu. Rev. Plant Physiol. 29, 437-460). In order to verify this important physiological role, a purified preparation of the yeast plasma membrane ATPase has been reconstituted with soybean phospholipids by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed a 32Pi-ATP exchange partially sensitive to proton ionophores (uncouplers) and to the proton-potassium exchange carrier nigericin. The reaction was completely inhibited by the nonspecific ionophore gramicidin and by the combination of uncouplers with the potassium ionophore valinomycin. These results are interpreted as evidence for two types of proton transport catalyzed by the enzyme preparation: electrogenic proton transport and electroneutral proton-potassium exchange.