To examine the functional properties of the three major isoforms of plasma membrane H⁺-ATPase expressed in Arabidopsis thaliana (AHA1, AHA2, and AHA3), we employed a system for the heterologous expression of functional plant plasma membrane H⁺-ATPase in yeast (Villalba, J. M., Palmgren, M. G., Berberian, G. E., Ferguson, C., and Serrano, R. (1992) J. Biol. Chem. 267, 12341-12349). Each isoform was expressed efficiently but appeared to be retained in the endoplasmic reticulum of yeast. All isoforms displayed qualitatively similar enzymatic properties, but quantitative differences were found. When compared with AHA3, AHA1 and AHA2 had an apparent higher turnover rate for ATP hydrolysis, exhibited a 10-fold higher apparent affinity for ATP, and a 3-fold higher sensitivity toward vanadate. In addition, AHA2 had a slightly lower apparent affinity for H⁺ and seemed to be more susceptible to activation by lysophosphatidylcholine than did AHA1 and AHA3. This study represents the first comparison of the functional properties of isoforms of the plant plasma membrane H⁺-ATPase.