Differential centrifugation and density gradient isopycnic centrifugation have been used to fractionate homogenates of rat spleen and, in a few experiments, of rat thymus and cervical lymph nodes. The fractions have been analyzed for proteins, DNA, RNA, cytochrome oxidase, esterase, and up to 11 acid hydrolases. The results obtained indicate that the hydrolases are associated, at least largely, with cytoplasmic particles of lysosomal nature, and suggest further that these particles belong to two, and possibly three, distinct populations, perhaps reflecting the cellular heterogeneity of the tissues. The populations are identified as: (a) the L19 population, the most important group, containing all 12 hydrolases and characterized by a modal density of about 1.19 in a sucrose—0.2 M KCl gradient; (b) the L15 population with a modal density of 1.15, a group of apparently incomplete lysosomes containing cathepsin D and a few other enzymes, but very poor in, or entirely devoid of, several acid hydrolases, including cathepsins B and C; (c) the L30 population, comprising all 12 enzymes and banding together with the nuclei at a density of 1.30 or higher. Lack of success in separating the latter group from the nuclei renders its significance unclear.

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