The separation of functional early and late endosomes from other cellular compartments by free-flow electrophoresis (FFE) has been previously demonstrated in nonpolarized cells [1, 2]. Here, using 125I-labeled anti- secretory component antibodies ([125I]SC Ab) and FITC-labeled asialoorosomucoid (FITC-ASOR) as markers of the transcytotic and lysosomal pathway, respectively, we demonstrate the separation of three distinct endosome subpopulations from polarized rat hepatocytes. Internalization of both markers at 16°C resulted in their accumulation in a common endosome compartment, indicating that both the transcytotic and the lysosomal pathways are arrested in the sorting early endosome at temperatures below 20°C. After chase of the markers from early endosomes into the transcytotic or the degradative route at 37°C, transcytotic endosomes carrying [125I]SC Ab migrated with an electrophoretic motility between early and late endosomes while late endosomes labeled with FITC-ASOR were deflected more towards the anode than early endosomes. These data indicate that in rat hepatocytes, the transcytotic and lysosomal pathways utilize a common (i.e. early endosomes) and two distinct endosome subpopulations (i.e. transcytotic endosomes, late endosomes) prior to delivering proteins for biliary secretion or lysosomal degradation, respectively.