Intravenously administered (125)I-labeled human beta-hexosaminidase A (beta-N-acetylglucosaminidase; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was rapidly cleared from the circulation of rats and accumulated in the liver. When hepatic cells were subsequently isolated, the label was recovered from both sinusoidal cells and, to a lesser extent, hepatocytes. Clearance was inhibited by the simultaneous infusion of mannan but not by a galactose-terminated glycoprotein. Studies in vitro, in which (125)I-beta-hexosaminidase was incubated with isolated hepatic cells, detected no uptake of the labeled ligand by hepatocytes. In contrast, uptake by sinusoidal cells was shown to be temperature dependent and approached saturability. Prior treatment of sinusoidal cells with Pronase resulted in markedly decreased uptake of (125)I-beta-hexosaminidase by these cells. Mannan and partially deglycosylated glycoproteins bearing terminal nonreducing N-acetylglucosamine or mannose residues were shown to be potent inhibitors of the cellular uptake of (125)I-beta-hexosaminidase; native orosomucoid and desialylated (galactoseterminated) orosomucoid were not inhibitory. Of six simple sugars tested, including N-acetylglucosamine, only mannose was an effective inhibitor of the cellular uptake of (125)I-beta-hexosaminidase. The kinetics of uptake of beta-hexosaminidase and mannose-terminated orosomucoid by sinusoidal cells were shown to be similar. These findings suggest that the hepatic uptake of the lysosomal glycosidase beta-hexosaminidase A is mediated by a receptor on sinusoidal cells which recognizes and binds mannose-terminated glycoproteins.