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The hydrolytic activity usually associated with lysosomes increased in the homogenates and subcellular fractions of rat liver as a result of essential fatty acid (EFA) deficiency. The proportion of the total (tissue homogenate) activity found in each subcellular fraction, however, was unchanged by EFA deficiency.Lysosomes isolated from normal and EFA-deficient rat livers differed significantly in their stability to thermal and osmotic variations. This suggested that lysosomal membranes, like other membranes, were altered by EFA deficiency.In spite of increased tissue-bound hydrolytic activity and altered lysosomal membranes, hydrolytic activity of the serum was not markedly changed in EFA deficiency. These minor changes in hydrolytic activity and in lysosomal membrane stability seemed insufficient to explain the general lesions of EFA deficiency.