Flavonoids play an important role as signal molecules in the early stages of symbiosis between legumes and Bradyrhizobium or Rhizobium. The flavonoid content of root exudates of individual seedlings of Glycine max cv. Preston was analyzed by a sensitive method that combines the adsorption of flavonoids on cellulose acetate filter strips and their separation by nano thin-layer chromatography plates, with their identification and quantification by an absorption scanner. B.japonicum 110spc4, which is able to form nitrogen-fixing nodules with this legume, caused a significant increase in daidzein, coumestrol, and genistein levels in root exudates of infected seedlings. The flavonoid accumulation can be attributed to the presence of suitable rhizobial lipooligosaccharide signals, called Nod factors. This conclusion is based on the following results: 1) B. japonicum Δ1240, a Nod^- mutant, lacking nodD₁D₂YABCS and therefore unable to produce Nod factors, failed to induce flavonoid accumulation. 2) Enhanced levels of daidzein, coumestrol, and genistein were also observed after G. max seedlings were treated with pure Nod factor of B. japonicum in nanomolar concentrations. 3) A similar flavonoid accumulation was induced by a nonsulfated Nod factor of Rhizobium sp. NGR234, another soybean-nodulating bacterium. Comparative assays with three Nod factors of NGR234, differing in the substitution of the 2-0-methylfucose residue, suggest that in the symbiosis with G. max the absence of the sulfate group is essential for the specific induction of flavonoid accumulation. Our data further develop the interaction model involving flavonoids and Nod factors as components of the signal chain between microsymbionts and legume host plants.