In polarized cells, the delivery of numerous membrane proteins from the trans Golgi network to the basolateral surface is dependent upon specific sequences located in their cytoplasmic domain. We have previously shown that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) exhibits a polarized cell surface distribution in the human colon adenocarcinoma cell line, Caco-2, in which there is a three-fold enrichment on the basolateral surface. To investigate the role of residues in the cytoplasmic region of the receptor that facilitate its entry into the basolateral sorting pathway, we have generated stably transfected Caco-2 cell lines expressing various mutant bovine IGF-II/MPRs. The steady state surface distribution of the mutant receptors was analyzed by subjecting filter-grown cell monolayers to incubation with iodinated IGF-II/MPR-specific antibody or to indirect immunofluorescence and visualization by confocal microscopy. Taken together, these results demonstrate that the sorting of the IGF-II/MPR to the basolateral cell surface depends upon recognition of sequences located in its cytoplasmic region that are distinct from the Tyr-based internalization and dileucine-dependent endosomal trafficking motifs.