Vacuoles can be isolated by flotation from suspensions of lysed proto-plasts in the presence of Ficoll. The purity of the respective preparations is demonstrated by the complete absence of enzymes of the mitochondria and of the groundplasm, as well as by morphological observations. Hydrolytic enzymes (two acid proteases, p-nitrophenylacetate-esterase, RNase and leucylaminopeptidase) are present in high specific activities in isolated vacuoles. The vacuole therefore represents the lysosome of the yeast cell.