The plant plasma membrane (PM) is the outermost membrane of the cell and constitutes the cell border across which nutrients are imported and metabolic products exported. It is also the site for receptors recording the environment and the interphase between the cell wall and the cytoskeleton. This makes the PM a key membrane for cell functioning and explains the great interest in its characterization. However, as the PM only constitutes 5-20% of the total membranes of a plant cell, a strong demand is placed on the isolation procedure to obtain pure PM. Furthermore, for studies on transport, there is a need for vesicles of defined sidedness, i.e., PM preparations where the vesicles expose either the apoplastic side (right-side out vesicles) or the cytoplasmic side (inside out vesicles) of the membrane to the medium. These vesicles should also be sealed to allow gradients to build up across the membrane. In this chapter, we describe how the PM is separated from all intracellular membranes resulting in a PM preparation with high yield and purity (1 ), and consisting of mainly sealed, right-side out vesicles (2 ). We also describe how these right-side out vesicles are everted to produce tightly sealed inside-out vesicles (3 ,4 ).