Microsomal preparations of six species of the plant family Fabaceae were screened for high-affinity binding of branched (1 → 3), (1 → 6)-β-glucans. Oligoglucosides of this type are specific elicitors of phytoalexin accumulation in soybean (Glycine max L.), a member of this family. The species studied were alfalfa (Medicago sativa L.), broadbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A 125I-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 → 3), (1 → 6)-β-glucan fraction with an average degree of polymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural complexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 → 3), (1 → 6) hepta-β-glucoside for which soybean binding sites display highest affinity. Radioligand competition experiments against unlabeled fungal β-glucan resulted in the identification of high-affinity binding in alfalfa, bean, lupin, and pea. Half-maximal competition concentrations (IC50) for fungal β-glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled hepta-β-glucoside showed that for alfalfa, lupin and pea the IC50 values for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC50 significantly higher than soybean (68 nM) suggesting that the structural motif recognized by its binding sites is not identical to that of soybean or the other three species. Radioligand saturation assays with alfalfa, lupin and pea microsomes using an 125I-labeled aminophenylethylamine hepta-β-glucoside conjugate gave dissociation constants (Kd) of 5.3, 3.7, and 1.8 nM, respectively. The affinity of these sites for hepta-β glucoside was in the same range as that of soybean (Kd 1–3 nM), whereas the affinity of the binding sites of bean for the same ligand was significantly lower (Kd = 33 nM). Good correlation was found between the presence of high-affinity binding and the accumulation of isoflavonoid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal β-glucan. Lupin displayed a strong wound-induced accumulation of prenylated isoflavones which was independent of the presence of β-glucan, making it impossible to determine phytoalexin induction in response to elicitor. No specific binding or phytoalexin accumulation in response to β glucans was observed in broadbean. This is the first report on the existence of possibly homologous elicitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathogen perception mechanisms in plants.