In earlier research we isolated and characterized three endosomal fractions from livers of estradiol-treated rats (1987. Belcher et al. Proc. Natl. Acad. Sci. USA. 84: 6785; 1989. Jäckle et al. Proc. Natl. Acad. Sci. USA. 86: 1880). We now describe the isolation of comparable endosomal fractions from untreated rats. The fraction of lowest density was composed almost exclusively of lipoprotein-filled multivesicular bodies (MVBs); the intermediate density fraction was composed of smaller lipoprotein-filled vesicles that were also multivesicular and had more numerous membranous appendages; the fraction of highest density was composed of membranes resembling the appendages of the two vesicular fractions. The paucity of contamination of these fractions with nonendosomal organelles is supported by their ultrastructural characteristics and by the proteins, lipids, and marker enzymes of their membranes. Only small amounts of MVBs could be separated from untreated rats not given a load of lipoproteins. However, after injection of large amounts of beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed rabbits, the mass of MVBs increased dramatically. Under these conditions radiolabeled beta-VLDL and epidermal growth factor taken up by the liver accumulated in isolated endosomes at rates similar to those found for LDL in estradiol-treated rats. Although chylomicrons and chylomicron remnants were rapidly taken up by the liver of normal rats, chylomicrons and chylomicron remnants accumulated in endosomes at a lower rate than beta-VLDL. These findings, which differ from earlier data in estradiol-treated rats (1989 Jäckle et al., Proc. Natl. Acad. Sci. USA. 86: 1880) that showed equivalent rates of processing of chylomicron remnants and beta-VLDL, suggest that extracellular processing of chylomicron remnants in the liver normally ; precedes endocytosis.