Nodulated (T202) and non‐nodulated (T201) isolines of soybean (Glycine max [L.] Merr.) were cultivated in a rotated paddy field in Niigata, Japan. The pods, and seeds were harvested at 7‐day intervals until maturity, and the subunit compositions of seed storage proteins were analyzed by SDS‐PAGE. The β‐subunit of β‐conglycinin could scarcely be detected in the non‐nodulated isoline, T201, at any period throughout seed development, although it was a major component in T202. The accumulation of α′‐ and α‐subunits of β‐conglycinin, together with the acidic and basic subunits of glycinin, appeared about one week later in seeds of T201 than in those of T202, perhaps due to a shortage of nitrogen and growth retardation. Northern hybridization could not detect the β‐subunit mRNA in immature T201 seeds, while it was pronounced in T202. These results indicate that the suppression of the β‐subunit in the non‐nodulating isoline T201 is regulated at the level of mRNA accumulation. The α′(α)‐subunit mRNAs were actively expressed in both isolines. Total nitrogen concentration was consistently lower in T201 than T202. No significant difference was observed in either the free amino acid or ureide concentrations in seeds, although the concentration of sucrose was considerably lower in T201 seeds and pods compared with T202. This result indicates the possibility that β‐subunit accunmlation was regulated not only directly by total nitrogen concentration but also by carbohydrate concentrations.Nitrogen regulation of storage protein subunit levels of soybean seed were evaluated using T201 and T202. Greenhouse‐grown plants were subjected to different levels and timing of nitrate treatments. The culture solution (2, 5 or 10 mM NO3–was supplied from flowering, 42 days after planting (DAP), until maturation (137 DAP), or switched from 2 to 10 mM, or from 10 to 2 mM at 61 DAP. With a continuous 2 mM NO3–treatment, seed dry weight and N concentration of the T201 plants were significantly lower than those in the T202 plants due to the lack of N2 fixation by the non nodulated T201 plants. However, when adequate NO3 was supplied, N concentration and dry weight were similar in T201 and T202 seeds. When 5 mM NO3 was supplied, the subunit proportion of the seed storage protein was similar in non‐nodulating and nodulating isolines. On the other hand, when plants received a low level of NO3 (2 mM), the β‐conglycinin proportion was lower in T201 than in T2O2. Furthermore, in the nodulating T202 plants treated with 10 mM NO3–the proportion of β‐conglycinin increased markedly.The results indicate that non‐nodulated T201 has a normal, non‐defective, β‐subunit gene and that limited N availability decreases accumulation of β‐conglycinin, whereas high N availability increases the proportion of β‐conglycinin in soybean seeds, irrespective of whether N was derived from N2 fixation or from NO3 absorption.