The nodule number of leguminous plants is controlled by “autoregulation”, a type of systemic regulation. Although many studies have attempted to identify key substances in this mechanism, none have been identified to date. Previously, we reported that the key substance(s) in this interaction are shoot-synthesized nodulation restricting substance(s) (SNRS), which are contained in the ethanol-soluble medium-polarity fraction of the shoot extract of wild-type soybean cv. Williams82. In the present study, extracts of wild-type soybean were separated by high-performance liquid chromatography (HPLC) and SNRS activity was detected in two different HPLC peaks (P-1, P-2) of the ethanol-soluble fraction. The P-1 and P-2 peaks were also found in the phloem sap of wild-type Williams82, but not in the sap of the hyper-nodulation mutant NOD1-3. A comparison of ultraviolet spectra implied that P-1 and P-2 do not contain salicylic acid, methyl jasmonate, abscisic acid, spermine or spermidine. Our results suggest that P-1 and P-2 contain two or more SNRS(s), although the purity of peaks P-1 and P-2 is still unclear.