Vacuoles were released from oat (Avena sativa) mesophyll protoplasts and purified by sedi­mentation and flotation. Disruption of isolated vacuoles followed by density gradient centri­fugation gave two membrane bands which after combination were further purified on sucrose gradients. A significant contamination by microbodies, thylakoids, mitochondria, endoplasmic reticulum and Golgi membranes can be excluded, whereas markers for plasma membrane and chloroplast envelope were present in the final membrane preparation.In the purified membrane fraction the following enzymatic activities were detected: NADH- cytochrome C reductase E.C. 1.6.2.2, ATPase E.C. 3.6.1.3. UDPG: sterol glucosyltransferase, UDP-Gal: diacylglycerol galactosyltransferase E.C. 2.4.1.46, glucan synthetase II, CDP-choline: diacylglycerol phosphocholinetransferase E.C. 2.7.8.2, formation of acylgalactosyl diacylglycerol and acyl-CoA thioesterase E.C. 3.1.2.2. None of these can be considered to be specific for the tonoplast. Acid phosphatase E.C. 3.1.3.2 was present in the cell sap.The vacuolar membranes contain phospholipids and glycolipids of the most complex composi­tion found so far in a membrane system isolated from mesophyll protoplasts. About half of the glycolipids were accounted for by glycosyl diacylglycerols usually considered to be confined to plastids. Steryl glycosides and acyl steryl glycosides were other prominent glycolipids. A cerebroside was the predominating lipid component of this membrane preparation.