Plasma membrane H+-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H+-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H+-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins. ; Background: The plasma membrane H + -ATPase generates electrochemical gradients in plants and fungi. The minimal subunit organization required for activity is not known. Results: We developed a protocol for reconstitution of active H + -ATPase in nanodiscs. Conclusion: The minimal functional unit of the H + -ATPase is a monomer. Significance: The plasma membrane H + -ATPase functions like well characterized cation pumping P-type ATPases. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.