The clover root weevil, Sitona lepidus, frequently feeds on N2 fixing rhizobial root nodules of white clover (Trifolium repens), which may contain isoflavonoids with defensive and plant regulatory properties. This study investigated the isoflavonoids present in N2 fixing (active) root nodules, root nodules that were not fixing N2 (inactive), and roots without nodules, and tested the behavioral responses of neonatal S. lepidus larvae to aglycones of the identified compounds. Formononetin concentrations were higher in the active nodules compared with inactive nodules and roots alone. Moreover, there was a statistically significant attraction to formononetin by S. lepidus in arena experiments, whereas the other isoflavonoids were unattractive. It is suggested that S. lepidus may have become tolerant to the toxic effects of formononetin with repeated exposure, and that it may play a role in root nodule location. Such coevolutionary relationships are widely reported for aboveground insects and plants, but the present study suggests they may also occur belowground.