We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.