Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.