The recognition of cell-surface L-selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of inflammation. As this primary contact is a prerequisite for extravasation of the leukocytes to the tissue, its inhibition by free oligosaccharides capable of competing with the natural L-selectin ligands in an attractive therapeutic possibility. The exact structures of the biological ligands of L-selectin are not yet known, but the principal carbohydrate epitopes share some structural features: they are O-glycosidically linked mucin-type oligosaccharides with N-acetyllactosamine backbone, which is 3'-sialylated or 3'-sulfated, 3-fucosylated and sometimes 6- or 6'-sulfated at the distal N-acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved by a branched polylactosamine backbone or by a clustered array of O-glycans. We report here enzymic synthesis of a large oligosaccharide fulfilling several of the features characteristic to the L-selectin ligands: it is a dodecameric O-glycosidic core-2-type oligosaccharide alditol with a branched polylactosamine backbone carrying two distal alpha-2,3'-sialylated and alpha-1,3-fucosylated N-acetyl-lactosamine groups (sialyl Lewis x, sialyl Le(x)). The structure of each saccharide on the synthesis route from disaccharide Gal beta 1-3GalNAc to the dodecasaccharide alditol was established by several methods including one- and two-dimensional 1H-NMR spectroscopy. The last step of the synthesis, the alpha-1,3-fucosylation of the 6-linked arm proceeded sluggishly, and was associated with a noticeable shift in H1 resonance of the GlcNAc residue of the branch-bearing N-acetyllactosamine unit. The final synthesis product and its analogs lacking one or both of the fucose residues were tested as inhibitors of L-selectin-mediated lymphocyte-endothelium interaction in vitro in rejecting rat kidney transplant. While the non-fucosylated O-glycosidic oligosaccharide alditol did not possess any inhibitory activity, the mono-fucosylated one (i.e. monovalent sialyl Le(x)) prevented the binding significantly and the difucosylated dodecasaccharide alditol (i.e. divalent sialyl Le(x)) was a very potent inhibitor (IC50, inhibitory concentration preventing 50% of binding = 0.15 microM). Besides the multivalency, also the Gal beta 1-3GalNAc-ol sequence of the O-glycosidic core appeared to increase the affinity of the glycan to L-selectin. This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylactosamine decasaccharide, similar to the divalent dodecasaccharide alditol, but lacking the reduced O-glycosidic core, was a less effective inhibitor (IC50 = 0.5 microM) than the O-glycosidic dodecasaccharide alditol.