Rat hepatocytes were isolated by perfusion with a Ca2+ free solution, followed by a buffer containing Ca2+ and collagenase. They specifically and avidly bind 125I-asialo-orosomucoid. At 4 degrees C, cells in minimal essential medium, incubated in the presence of saturating concentrations of 125I-asialo-orosomucoid, bound 500,000 molecules/cell. Addition of 10% horse serum to the binding reaction reduced the number bound to 200,000. Prewashing of the cells in 1% bovine serum albumin reduced the number of bound molecules by 60%. In all cases, the Kd value was approximately 7 x 10(-9) M. At 37 degrees C, the rate of uptake of 125I-asialo-orosomucoid (approximately 0.1 pmol/min/10(6) cells) is proportionately reduced by the presence of serum or albumin preparatons. Thus, it appears that the absolute number of asialoglycoprotein receptors on the surface of rat hepatocytes is not readily determined in the presence of serum or albumin and is about 500,000/cell.