Hepatocytes rapidly internalize epidermal growth factor (EGF) and transferrin by receptor-mediated endocytosis. Both EGF and its receptor are thought to be targeted for destruction in lysosomes, leading to down-regulation of the receptor, whereas transferrin, after unloading iron within the cell, is thought to recycle to the cell surface bound to its receptor. Previously, we isolated three endosomal fractions from livers of estradiol-treated rats and examined their roles in cellular trafficking of low density lipoproteins (LDL) and the LDL receptor, which cycles constitutively (Belcher, J. D., Hamilton, R. L., Brady, S. E., Hornick, C. A., Jäckle, S., Schneider, W. J., and Havel, R. J., Proc. Natl. Acad. Sci. U. S. A. (1987) 84, 6785-6789). In the current study we have taken advantage of the distinct trafficking of the EGF receptor and transferrin to evaluate further the functions of these endosome fractions. Intravenous injection of a saturating amount of EGF into estradiol-treated rats induced internalization of a single population of EGF receptors, which rapidly accumulated in the endosome fraction of intermediate density ("compartment of uncoupling of receptor and ligand" (CURL)) and subsequently in the low density endosome fraction (multivesicular bodies (MVBs)). The high density endosome fraction, whose membranes contain a high concentration of recycling receptors (designated receptor-recycling compartment (RRC)), failed to accumulate EGF receptors after injection of EGF. In livers of rats not given exogenous EGF, EGF receptors were found in small but comparable concentrations in RRC, CURL, and MVB membranes, consistent with other evidence that targeting of the EGF receptor to lysosomes is mediated by ligand-induced phosphorylation. Transferrin also accumulated first in CURL and later in MVBs, but it also accumulated rapidly in the RRC fraction, consistent with the proposed function of this fraction in receptor recycling. Since transferrin is not degraded during its endocytic cycle, these observations indicate that apotransferrin and its receptor recycle from late endosomes (MVBs) located at the apical pole of hepatocytes, as well as from early endosomes near the sinusoidal pole.