Rat hepatocytes prepared by collagenase perfusion of the liver are known to exhibit increased binding of asialoorosomucoid (ASOR) after prior treatment with EDTA or after warming at 37°C. The cause of the apparent increase in the surface binding activity of the galactose/N-acetylgalactosamine (Gal/GalNAc) receptor on freshly isolated rat hepatocytes was investigated. Binding experiments using three different galactose-terminated ligands revealed up to a 2-to 6-fold increase in the level of surface receptor sites on rat hepatocytes upon prior incubation at 4°C with 10 mM GalNAc or 10 mM EDTA or at 37°C compared to untreated cells. With digitonin-permeabilized cells, it was shown that the newly exposed receptor sites most likely originated from masked surface receptor sites, as no alteration in the size of the internal pool of receptor was observed. Collagenase preparations were found to inhibit the binding of 121I-labeled ASOR to the Gal/GalNAc receptor. Exposure of hepatocytes to collagenase resulted in a significant decrease in 12.5-labeled ASOR binding, which was reversible upon treatment with GalNAc or EDTA at 4°C or upon warming at 37°C. Perfusion of EDTA through the isolated whole liver at 0-20C to remove any possible bound endogenous ligands did not result in a significant increase in the level of 1251-labeled ASOR binding, while perfusion of collagenase caused a marked decrease in the binding activity of the liver. We conclude that the enhancement of Gal/GalNAc receptor activity on the surface of freshly isolated hepatocytes by temperature and EDTA is potentially an artifact of the collagenase perfusion method.The galactose/N-acetylgalactosamine (Gal/GalNAc) receptor of the parenchymal cells of mammalian liver functions in the binding and internalization of galactose-and GalNActerminated oligosaccharides and glycoproteins (for review, see refs. 1-4). This receptor system has been extensively studied in both the isolated perfused liver (5) and in isolated hepatocytes prepared by a two-step collagenase-perfusion technique (6) with the goal of elucidating the pathway and molecular mechanisms of receptor-mediated endocytosis. The first step in the pathway of Gal/GalNAc receptormediated endocytosis is the specific binding of ligand to receptors on the plasma membrane of hepatocytes. In the case of freshly isolated rat hepatocytes, there is considerable variability in the literature regarding the surface binding behavior of asialoorosomucoid (ASOR), particularly in the magnitude of the receptor site number. The sources of variability in site numbers for this receptor are potentially numerous with regard to the conditions of the collagenase perfusion protocol and the binding assay and have been acknowledged (7). In addition, several treatments have been reported to affect the expression of surface receptor activity in this system and include temperature (8-12) and EDTA (9,13).In this paper we present evidence that collagenase preparations contain a carbohydrate-containing inhibitor(s) that is specificall...