Results of grafting experiments between super-nodulation (or hyper-nodulation) mutants of soybean and their parents reconfirmed that super nodulation is a shoot-controlled phenomenon, suggesting that a systemic regulatory mechanism acts in soybean plants and a specific nodulation-controlling substance (SNS) is synthesized in the shoot and transported to the roots. To search for the SNS involved in the super-nodulation trait of NOD1-3, a mutant of soybean (Glycine max [L.] Merr. cv. Williams), we adopted a bioassay system using plantlets derived from the first trifoliate leaf of the seedlings; this system enabled us to introduce liquid substances continuously into leaves and to assess their effect on root nodulation. Following the application of leaf extract from Williams82 plants lacking visible root nodules, formation of root nodule meristems in NOD1-3 plantlets was repressed on the sixth day after rhizobial inoculation and the number of visible nodules on the eighth day declined to the same level as that in the Williams82 plantlets. Application of NOD1-3 leaf extract resulted in no significant change in the nodulation of both NOD1-3 and Williams82 plantlets. These results suggested that the SNS is a downregulator of nodulation and is responsible for the wild-type (Williams82) phenotype, and that the super-nodulation phenomenon is caused by a paucity of the SNS. The intensity of the repressive effect of the Williams82 leaf extract was not changed by nodulation of the source plants, thus we conclude that visible nodule formation is not required to induce production of the SNS.