Exposure of oat seedlings to repeated moderate water deficit stress causes a drought acclimation of the seedlings. This acclimation is associated with changes in the lipid composition of the plasma membrane of root cells. Here, plasma membranes from root cells of acclimated and control plants were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracted from the plasma membranes. In a series of tests the vesicle permeability for glucose and for protons were analysed and compared with the permeability of model vesicles. Further, the importance of critical components for the permeability properties was analysed by modifying the lipid composition of the vesicles from acclimated and from control plants. The purpose was to add specific lipids to vesicles from acclimated plants to mimic the composition of the vesicles from control plants and vice versa. The plasma membrane lipid vesicles from acclimated plants had a significantly increased permeability for glucose and decreased permeability for protons as compared to control vesicles. The results point to the importance of the ratio phosphatidylcholine (PC)/phosphatidylethanolamine (PE), the levels of cerebrosides and free sterols and the possible interaction of these components for the plasma membrane as a permeability barrier.