Carbohydrate-deficient Glycoprotein Syndrome (CDGS) is an autosomal recessive congenital disorder that affects many organ systems. It is characterized by specific physical stigmata as well as hypotonia, retardation, hepatopathy, and pericardial effusion. The basic cellular defect is supposed to be a cotranslational asparagine-N-linked oligosaccharide transfer deficiency in the endoplasmic reticulum (ER) (Yamashita et al., 1993, J. Biol. Chem. 268 : 5783-5789). The cotranslational oligosaccharide transfer is crucial for correct folding of many newly synthesized glycoproteins. If it is inhibited by tunicamycin treatment or mutation of the glycosylation site these proteins experience severe problems in their folding process leading to irreversible aggregation and retention in the ER. By electron microscopy we found that in CDGS fibroblasts the ER is dilated indicating a possible retention phenomenon. For a more detailed analysis several well characterized viral glycoproteins were expressed in these cells (influenza hemagglutinin, SFV spike glycoproteins). Their glycosylation and folding was investigated by immunoprecipitation with specific antibodies and subsequent analysis by non-reducing SDS-PAGE. We found that cotranslational glycosylation and folding of these model glycoproteins were undisturbed. Endoglycosidase treatments of the samples revealed that the transport to the Golgi was delayed. Our findings demonstrate that in CDGS the cell biological defect is located at the level of the endoplasmic reticulum. However, they contradict the current hypothesis of a cotranslational glycosylation defect.