In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysinebased oligomannosides containing two (M 2 L) to six (M 6 L 5 ) terminal ␣-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 Å). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissuetype plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18 -23 M to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional ␣-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M 3 L 2 to M 6 L 5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M 6 L 5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M 6 L 5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.The mannose receptor is a 175-kDa membrane-associated protein that is localized on sinusoidal liver cells, peripheral and bone marrow macrophages, and dendritic cells (1-4). It recognizes and internalizes mannosylated polysaccharides from pathological microorganisms (5), tumor cells (6), and yeast cells (7) and glycoproteins like type-I procollagen (8), tissue-type plasminogen activator (9), or various lysosomal enzymes (10). As such, the mannose receptor participates in the nonimmune host-defense system. In addition, the macrophage receptor is implicated in major histocompatability complex-mediated antigen presentation by dendritic cells (11).The cDNA of the mannose receptor has been sequenced by Taylor et al. (12) and codes for five types of domains (13): an N-terminal cysteine-rich domain, a transmembrane domain, a fibronectin type II-like domain, a domain composed of eight strongly homologous repeats (the so-called carbohydrate recognition domains or CRDs)1 and a C-terminal cytoplasmic tail. Taylor and Drickamer (13, 14) have established that the CRDs are involved in ligand binding. Recent structure-function studies of recombinant truncated forms of the mannose receptor provided new insight into the mechanism of ligand binding by the mannose receptor (13,14). On basis of these results, it was proposed that CRD4 is the only CRD to display a monosaccharide specificity characteristic for the mannose receptor (15). CRD4 and CRD5 appear to be required for high affinity binding of high mannose-type glycoproteins and mannosylated bovine serum albumin (BSA). B...