Abstract: Using the asialoglycoprotein receptor (ASGP-R) and a representative ligand, asialoorosomucoid (ASOR), we have previously shown ethanol-induced impairment of endosomal acidification, receptor recycling and ligand binding, internalization, and degradation. In the current study, we further investigated ethanol-induced alterations in receptor/ligand trafficking by labeling endosomes in vivo with either Texas-Red-ASOR or 125I-ASOR, and then assessing the receptor/ligand content of endosomes. We assessed two fractions after both 5 and 25 min of labeling: ‘early endosomes’ (EEs; endosomes from the cell periphery) and ‘late endosomes’ (LEs; endosomes farther into the cell interior). At both time points, significantly more ligand was found in EE fractions isolated from chow- and pair-fed controls (3:1, EE to LE, respectively). However, endosomes isolated from ethanol-fed animals showed a shift over time toward a more equal ligand distribution between endosome fractions (P≤0.05). Analysis of the ASGP-R content revealed a distribution pattern between the endosome fractions similar to that observed for ligand distribution. Impairment of receptor–ligand dissociation was assessed in endosome fractions by determining bound/free ligand ratios. Analysis showed that most of the ligand present in both endosome fractions was free (56–99%), although more was bound to receptor in EE vs LE of both control and ethanol animals (P≤0.05). At 5 min, more ligand remained bound in endosomes from ethanol-fed animals compared with control endosomes (P≤0.05), and the same pattern was observed at the latter time point. These results suggest that delayed dissociation may cause the receptor–ligand complexes to travel farther into the cell interior, which may impair proper trafficking of the ligand to lysosomes and alter the receptor recycling.