Abstract. The sorting of macromolecules within and between membranous organelles is often directed by information contained in protein primary or secondary structure. We show here that absent such structural information, macromolecules internalized by endocytosis in macrophages can be sorted by size. After endocytosis, small solute probes of fluid-phase pinocytosis were recycled to the extracellular medium more efficiently than large solutes. Using macropinosomes pulse labeled with fluorescent dextrans, we examined the ability of organelles to exchange solute contents. Dextran exchange was optimal between organelles of similar age, and small dextrans exchanged more efficiently than large dextrans. Efferent solute movement, from lysosomes or phagolysosomes toward the plasma membrane, occurred through the same endocytic vesicles as afferent movement, toward lysosomes and this movement was solute size dependent. Remarkably, uniform mixtures of different-sized dextrans delivered into lysosomes separated into distinct organelles containing only one dextran or the other. Thus, the dynamics of endosomes and lysosomes were sufficient to segregate macromolecules by size. This intracellular size fractionation could explain how, during antigen presentation, peptides generated by lysosomal proteases recycle selectively from lysosomes to endosomes for association with class II MHC molecules.T HE traffic of macromolecules within and through vesicular organelles is directed both by the dynamics of the organelles and by information contained in the structure of the macromolecules. Light and electron microscopic studies of endocytic organelle dynamics indicate a high degree of communication between endosomes and lysosomes, mediated by vesicle fusion and fission and by microtubule-mediated organelle movements. In most cells, solute internalized by fluid-phase endocytosis moves sequentially through early endosomes and late endosomes into lysosomes, with some percentage of that solute leaving the cell via vesicular recycling. Macromolecules that take other paths carry information in their structure that enhances either retention in endosomes or movement into other compartments. Such sorting information is often contained in protein primary or secondary structure, and works by selectively directing molecules into transport vesicles or by mediating associations with other molecules that are so directed (Sandoval and Bakke, 1994). Some molecular sorting may occur without such signals, however. As antigen-presenting cells, macrophages process many different and essentially unrecognized proteins. Proteins delivered to lysosomes via endocytosis are degraded to peptides, which then recycle to endosomes or to other nonlysosomal compartments, where they associate with MHC class II molecules (Germain, 1994 lysosomes to endosomes indicates a sorting process, in that these small molecules are recycled but the lysosomal enzymes are not. The great variety of peptides that travel this route makes it unlikely that sorting information in their prim...