In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3 H]mannose-labeled Man 5 GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man 5 GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (K uptake , 22.3 M, V max , 7.1 fmol/min/unit of -hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H ؉ /ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.Free polymannose-type oligosaccharides (free OS) 1 are a byproduct of the biosynthesis of dolichol oligosaccharides (1-3) and the glycosylation of glycoproteins (3, 4). Whereas phosphorylated free OS (Man 5 GlcNAc 2 -P) are released from dolichol oligosaccharides on the cytoplasmic face of the ER (5), neutral free OS (Glc 3-1 Man 9 -8 GlcNAc 2 ) are liberated from oligosaccharide-lipid in the lumen of the ER (1, 6). After deglucosylation, these neutral free OS do not follow the secretory pathway but are routed to the cytosol (7) by an ATP-and calcium-requiring translocation process that can be inhibited by mannose but not by N-acetylglucosamine (8). Glycopepetides (9, 10) and misfolded glycoproteins (11,12) are also translocated out of the ER into the cytosol, where they can be deglycosylated by an Nglycanase (13, 14), giving rise to further cytosolic free OS.The rapid appearance of substantial quantities of free OS in the cytosol of cells during the biosynthesis and quality control of glycoproteins suggested that the cytosol must possess elements capable of the disposal of these osmotically active components. In fact, neutral free OS are subject to sequential processing (15-17) by a previously characterized cytosolic endo-H-like activity (18) or chitobiase (15) and cytosolic mannosidase (19 -21) to yield a free OS containing five residues of mannose and a ...