Desialylated glycoprotein is rapidly cleared from plasma by a receptor-mediated endocytic mechanism located on hepatocytes. We studied the hepatic acinar distribution of this asialoglycoprotein transport system with the ligand 125I-asialoorosomucoid using rat liver perfused in either antegrade or retrograde direction in combination with quantitative light microscopic autoradiography. Grain distribution along the acinus appeared dependent on the perfusion direction. A rather shallow zone 1 to zone 3 gradient was observed if livers were perfused in the normal direction. However, a statistically significantly steeper zone 3 to zone 1 gradient was detected in retrograde perfusions. Kinetic analysis of perfusate clearance profiles yielded a hepatic clearance of 21.6 ± 1.3 ml per min in antegradely perfused liver. Hepatic extraction was calculated to be 60.1 ± 7.4%. Biliary secretion of radioactivity amounted to 1.89 ± 0.18% of the dose within 1 hr after injection and consisted of intact material (1.39 ± 0.25%) and radioactive low-molecular-weight degradation products (0.52 ± 0.08%), of which more than 90% could be accounted for by 125I. Apart from a minor difference regarding biliary secretion of an unidentified glycopeptide (less than 0.1% of the injected dose), transport data for the retrogradely perfused livers were identical to those obtained with livers perfused in antegrade direction, emphasizing the functional equivalence of both groups of livers. The autoradiographic data indicate that zone 3 hepatocytes take up 125I-asialoorosomucoid more avidly than zone 1 cells. The kinetic and biochemical data indicate that further processing in the hepatocytes is virtually similar in the two zones. The functional significance of this higher uptake in zone 3 is presently unknown. A possible role in the mechanism of removal of senescent plasma proteins is discussed.