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MAIN CONCLUSION Variations in the content of tonoplast microdomains, isolated with the aid of a non-detergent technique, are induced by osmotic stress and may take part in plant cell adaptive mechanisms. Investigation of tonoplast microdomain lipids isolated with the aid of the non-detergent technique from beetroots (Beta vulgaris L.) subjected to either hyperosmotic or hypoosmotic stress was conducted. Earlier, an important role of tonoplast lipids in the protection of plant cells from stress was demonstrated (Ozolina et al. 2020a). In the present paper, we have put forward a hypothesis that lipids of microdomains of raft nature present in the tonoplast are responsible for this protective function. The variations in the content of lipids of the studied nondetergent-isolated microdomains (NIMs) under hyperosmotic and hypoosmotic stresses were different. Under hyperosmotic stress, in the scrutinized microdomains, some variations in the content of lipids were registered, which were characteristic of the already known protective anti-stress mechanisms. These variations were represented by an increase in sterols and polar lipids capable of stabilizing the bilayer structure of the membranes. The found variations in the content of sterols may be bound up with some intensification of the autophagy process under stress because sterols foster the formation of new membrane contacts necessary for this process. Under hypoosmotic stress, the pattern of redistribution of the lipids in the scrutinized membrane structures was different: the largest part of the lipids appeared to be represented by hydrocarbons, which fulfilled mainly a protective function in plants and could prevent the excess water influx into the vacuole. The results obtained not only demonstrate the possible functions of the vacuolar membrane microdomains but also put forward an assumption on the role of any membrane microdomain in the protection mechanisms of the plant cell.