The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.