*Legumes regulate the number of nodules they form via a process called autoregulation of nodulation (AON). This involves a shoot-derived inhibitor (SDI) molecule that is synthesized in the shoots and is transported down to the roots where it inhibits further nodule development. *To characterize SDI, we developed a novel feeding bioassay. This involved feeding aqueous leaf extracts directly into the petiole of hypernodulating and supernodulating nark mutant plants of Glycine max (soybean). These mutants normally exhibit an increased nodulation phenotype because SDI is not produced and thus AON is nonfunctional. *Feeding wild-type leaf extracts presumed to contain SDI was successful in suppressing the increased nodulation phenotype, whereas feeding with Gmnark leaf extracts did not. Suppression activity was inoculation-dependent, Nod factor-dependent, required GmNARK activity, and was heat-, Proteinase K- and ribonuclease A-resistant. Wild-type extracts maintained suppressive activity even at a ninefold dilution. Sinorhizobium meliloti-inoculated Medicago truncatula leaf extracts from wild-type, but not from supernodulating mutant Mtsunn, suppressed hypernodulation in soybean. *Our results demonstrate that the petiole feeding bioassay is an efficient and effective technique to introduce aqueous extracts into plants. They also demonstrate that SDI is a small compound with an apparent molecular mass of < 1000 Da and is unlikely to be a protein or an RNA molecule.