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Red beet (Beta vulgaris L., cv. Detroit Dark Red) plasma membrane ATPase solubilized from a deoxycholate-extracted plasma membrane fraction with Zwittergent 3-14 was reconstituted into liposomes. Detergent removal and reconstitution was carried out by column chromatography on Sephadex G-200 followed by centrifugation at 100 000 g for 1 h. Prior to reconstitution, optimal activity in the solubilized preparation was observed when dormant red beet tissue was used in the extraction/solubilization procedure. Following reconstitution into liposomes, ATP-dependent proton transport could be demonstrated by measuring the quenching of acridine orange fluorescence. Proton transport and ATPase activity in the reconstituted enzyme preparation were inhibited by orthovandate but stimulated by KNO3. This stimulation most likely results from a reduction in the membrane potential generated during electrogenic proton transport by the reconstituted ATPase. The ATPase activity of the reconstituted ATPase was further characterized and found to have a pH optimum of 6.5 in the presence of both Mg2+ and K+. The activity was specific for ATP, insensitive to ouabain and azide but inhibited by N',N-dicyclohexylcarbodiimide and diethylstilbestrol. Stimulation of ATP hydrolytic activity occurred in the sequence: K+ > Rb+ > Na+ > Cs+ > Li+ and the kinetics of K+ stimulation of ATPase activity followed non-Michaelis-Menten kinetics as observed for both the membrane-bound and solubilized forms of the enzyme. Reconstitution of the plasma membrane ATPase from red beet allowed a substantial purification of the enzyme and resulted in the enrichment of a 100 kDa polypeptide representing the ATPase catalytic subunit.