Previously, we reported that shoot-synthesized nodulation-restricting substance(s) (SNRS) was present in extracts from shoots of a wild-type soybean, cv. Williams82. In the present paper, we report SNRS activity of fractions prepared from shoot extracts of Williams82. Activity of the ethanol-soluble medium-polarity fraction (MPF), which was prepared by solid-phase extraction of the shoot extract using an octadecyl-bonded silica (ODS) cartridge, was similar to that of the shoot extract. A high-polarity fraction of the extract was separated into subfractions, which were tested for SNRS activity. The anionic plus non-ionic fraction and the cationic plus amphoteric fraction showed no SNRS activity. Our findings indicate that SNRS is present in the MPF of an extract prepared from shoots of Williams82.