The complex mixture of sterols, namely phytosterols, found in the plasma membrane (PM) of plant cells is an intriguing characteristic. In the present work, we characterized the sterol composition of tobacco BY-2 cell PM, and we adapted a sterol manipulation method to this plant material. Methyl-b-cyclodextrin was found to induce a drastic reduction of the PM total free sterol content, with a low specificity for the different phytosterols, and without effect on steryl-conjugates. The consequences of phytosterols depletion were investigated by means of fluorescence spectroscopy and biochemical detergent resistant membrane (DRM) fraction purification methods. Data obtained with the fluorescent probes DPH, TMA-DPH, Laurdand and di-4-ANEPPDHQ showed that lipid acyl chain order is reduced after free sterol depletion and suggested a diminution of the overall liquid-phase heterogeneity correlated with the disruption of phytosterol-rich domains. Finally, methyl-b-cyclodextrin treatment of tobacco PM prevented isolation of a DRM fraction. These findings clearly indicate the crucial role of phytosterols in the structuration of the plasma membrane of higher plant cells and particularly in the formation of lipid-rafts microdomains.