Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: Vitamin B12) and they obtain it from the circulation bound to transcobalamin (TC) via transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labeling, domain specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T1/2) of 8h following its delivery to the basolateral plasma membrane (BLM). The T1/2 of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T1/2, 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM.