OBJECTIVE This study demonstrates and characterizes apical (canalicular) endocytic pathways in hepatocytes in situ. METHODS Endocytic markers were administered by retrograde infusion through the common bile duct. Colocalization with proteins that are specific for various endocytic compartments was performed on stacks of deconvoluted confocal immunofluorescence images. The subcellular distribution of marker proteins was assessed by electron microscopy (EM). RESULTS Bulk-phase, as well as membrane-associated markers, were internalized readily at the apical cell pole. At the EM level, marker was found initially in 60-100-nm tubulovesicular structures and 150-200-nm cup-shaped vesicles, whereas multivesicular bodies and lysosomes became labeled after longer time intervals. Apical endocytosis involved clathrin and delivered marker to late endosomes (rab7(+), cathepsin D(+)), as well as lysosomes (rab7(-), cathepsin D(+)). Simultaneous labeling of the basolateral endocytic route resulted in overlap of both pathways in the late endosomal and lysosomal compartments. In addition, apical endocytosis involved a subapical compartment (endolyn-78(+), rab11(+), polymeric IgA receptor [pIgA-R(+)]) that is passed by the transcytotic route, thus constituting a crossroads. pIgA-R immunoreactivity, probably reflecting the cleaved receptor fragment, was associated with apical endocytic marker and colocalized with clathrin and later with cathepsin D. CONCLUSIONS Apical endocytosis involves coated pits/vesicles, leads to a subapical compartment, and plays a role in the retrieval of canalicular plasma membrane components for lysosomal degradation.