It has been postulated that the in vivo removal of many plasma glycoproteins after desialylation is mediated by their interaction with a specific endocytic receptor on hepatocytes called the asialoglycoprotein receptor (ASGP-R), which is known to have a high affinity for specific carbohydrate residues, such as galactose. However, this mechanism has never been proven in vivo, nor has a naturally occurring ligand for the ASGP-R been identified. We investigated the influence of the terminal galactose residues on plasma fibronectin (pFn) on its liver deposition and turnover in adult rats, using neuraminidase to remove sialic acid residues to expose galactose residues. We also tested the hypothesis that the normal presence of a large amount of terminal galactose residues in cellular Fn (cFn) may allow cFn to serve as a natural ligand readily able to interact with the ASGP-R. In contrast to the slow clearance of normal pFn from the blood, cFn and desialylated pFn (aFn) displayed a rapid plasma clearance (P < .001) with greater than 50% of both the 125I-cFn or 125I-aFn depositing in the liver within 15 minutes. The enhanced plasma removal and liver deposition of both 125I-cFn and 125I-aFn was competitively inhibited (P < .01) by prior intravenous infusion of excess asialofetuin, which can selectively bind to the ASGP-R. The enzymatic addition of terminal sialic acid residues onto cFn to “mask” or “cap” the normally exposed galactose residues delayed the rapid plasma removal of cFn. Accelerated degradation of 125I-aFn and 125I-cFn as compared with 125I-pFn was demonstrated in vitro by both primary cultures of normal rat hepatocytes or incubated (37°C) tissue slices of livers harvested from normal rats after in vivo preloading with tracer 125I-Fn forms. Thus, the ASGP-R appears to directly participate in the rapid in vivo removal of cFn from the blood, while native pFn may be removed by an alternative pathway unless it can become desialylated in vivo. These findings suggest that cFn may be a naturally occurring ligand that does not require desialylation before removal by the ASGP-R on hepatocytes.