Some putative key-substances translocated from leaves to roots were suggested to be involved in the supernodulation trait of NOD1-3, a mutant of soybean (Glycine max [L.] Merr.) cv. Williams. To search such key-substances, it is necessary to establish a bioassay-system which enables us to introduce substances continuously into leaves and to assess their effects on root nodulation. As a bio-assay system,.plantlets of 7-10 day-old cuttings were prepared using the first trifoliolate of soybean cv. Williams82 and NOD1-3, and applied to assess the effects of continuous introduction of 438 mM sucrose and 167 mM ^<15>N-labeled urea solutions on the formation of root-nodule meristem. The solutions were introduced into apoplast of vascular bundle of the plantlet leaf by self-suction via cut-surface of petiolule of the central foliolate. Root nodule meristems were microscopically inspected at the 5th day after rhizobium inoculation, which had been done after initiation of the continuous introduction. Urea introduction significantly repressed both emergence and development of root nodule meristem, especially in NOD1-3 plantlet. ^<15>N-analyses revealed that a significant amount of nitrogen originating from introduced urea was translocated to roots at the fifth day after initiation of ^<15>N-feeding. Sucrose introduction enhanced both emergence and development of root nodule meristem, irrespective-of the genotype or light condition. Conspicuous difference in root nodule formation between plantlets of NOD1-3 and Williams82 did never disappear under any conditions of exhaustion, normal or plentiful phtosynthate supply to plantlet roots. In our study, the liquids were successfully introduced into plantlets continuously for 5-7 days and some substances introduced into foliolates were expected to be translocatable to root. It was concluded that our bioassay system is applicable to searching for key substances involved in supernodulation phenomenon.