We have isolated and characterized the extracellular Nod factors of Rhizobium fredii USDA257, a nitrogen-fixing symbiont of soybean [Glycine max (L.) Merr.] and several other legume species. These signals are produced upon exposure to the isoflavone genistein and consist of a series of substituted, beta 1,4-linked tri-, tetra-, and pentamers of N-acetylglucosamine. N-Vaccenic acid replaces acetate on the nonreducing residue, and the reducing residue contains alpha-linked 2-O-methylfucose on carbon 6. Small amounts of a fucose-containing tetramer also were present. The Nod factors elicit root-hair deformations on soybean and two other plants at concentrations ranging from 10(-6) to 10(-12) M.