We have studied uptake of retinol-binding protein (RBP) by rat liver cells. First, we compared the in vivo uptake in different liver cells of 125I-labeled RBP with that of other well-known ligands. We found that the ligands studied were recognized differently by the various cell types in the liver, and that RBP was most efficiently taken up by parenchymal and stellate cells. We then studied the in vivo uptake of RBP in liver cells by immunocytochemistry at the electron microscopic level using ultrathin cryosections. Ten min after injection, RBP was localized to parenchymal cells and stellate cells. In these cells, RBP was detected on the cell surface and in vesicles near the cell surface. RBP was observed mainly in association with the membrane in these vesicles. Two hours after injection, RBP was localized not only on the cell surface and in vesicles close to the cell surface, but also in larger vesicles located deeper in the cytoplasm of these cells. RBP in larger vesicles was observed at a distance from the vesicular membrane. Finally, we compared the distribution of endocytosed RBP in liver parenchymal cells with that of asialo-orosomucoid, a ligand known to be internalized by receptor-mediated endocytosis. We detected both ligands on the cell surface and in small vesicles located close to the cell surface and in larger vesicles located deeper in the cytoplasm. Asialo-orosomucoid and RBP were seldom observed in the same small vesicles, but the larger vesicles contained both ligands. These data suggest that RBP is internalized in parenchymal and stellate cells of the liver by receptor-mediated endocytosis.