The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5'-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5'-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.