The establishment of the symbiosis between Rhizobia and the plants of the legume family depends on a signal exchange between the two partners, and shows a high degree of specificity determined largely by the bacterial signals called nodulation factors (Nod factors). Nod factors are able to induce in the host plant, at very low concentrations (10-12M) and in a structure dependent manner, many of the responses observed in the presence of the bacterium, suggesting the presence of receptors. By using radiolabelled Nod factors, we have been able to characterise in Medicago truncatula, a high affinity binding site (Kd = 0.45 nM) for the major Nod factor produced by its symbiont Sinorhizobium meliloti. This site termed Nod Factor Binding Site 3 (NFBS3) is associated to a high density fraction prepared from roots. Studies of Nod factor binding in root extracts of early symbiotic mutants of M. truncatula, reveals that NFBS3 is present in a nfp mutant, affected in a putative Nod factor receptor, but is absent in dmi1 and dmi2 mutants affected in genes required for the establishment of symbioses with both rhizobia and arbuscular mycorrhizal fungi. The potential role of NFBS3 in the establishment of root endosymbioses is discussed.