When grown in a nutrient solution containing combined nitrogen (NH4NO3), Lotus pedunculatus and L. tenuis seedlings inoculated with a fast-growing strain of Rhizoblum (NZP2037) did neither develop root nodules nor develop flavolans in their roots. In contrast, the roots of nodulated seedlings growing in a nitrogen-free nutrient solution contained flavolans. Flavolan synthesis coincided with root nodule development on these plants. When added as a single dose, high concentrations of NH4NO3 (5 and 10 mg N per plant) stimulated the growth of L. pedunculatus plants but suppressed nodulation and nitrogen fixation. In contrast the continued supply of a low concentration of NH4NO3 (1·0 mg N d−1 per plant) stimulated nitrogen fixation by up to 500%. This large increase in nitrogen fixation was associated with a large increase in nodule fresh weight per plant, a doubling of nodule nitrogenase activity, and a lowering of the flavolan content of the plant roots. The close relationship between nitrogen deficiency, nodule development, and flavolan synthesis in L. pedunculatus meant that it was not possible (by nitrogen pretreatment of plants) to alter the ineffective nodule response of a Rhizobium strain (NZP2213) sensitive to the flavolan present in the roots of this plant.