The intracellular site of uptake and degradation of globin-haptoglobin, the protein moiety of hemoglobin-haptoglobin, in rat liver cells was investigated in vivo. Hemoglobin-haptoglobin, administered intravenously to rats, is cleared from circulation and incorporated exclusively into liver parenchymal cells through the receptor specific for the molecule (Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H., and Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620). Intrahepatocellular distribution of radioactivity was determined after intravenous administration of (125I-hemoglobin)-haptoglobin or hemoglobin-(125I-haptoglobin) to rats. The 125I-labeled hemoglobin-haptoglobin was incorporated first in organelles of low density (density range, 1.05-1.07 g/ml) recovered in Golgi subfractions of the liver cells in a substantially intact form. The organelles progressively acquired a higher density, presumably through fusion with primary lysosomes. In the resulting organelles of high density (density range, 1.07-1.15 g/ml), which are probably secondary lysosomes, hemoglobin-haptoglobin first dissociated symmetrically to yield two 82,000-dalton subunits by a limited proteolysis, and further digestion of the constituent polypeptide chains seemed to proceed thereafter in the organelles during the transport process across the cells.