Aims: The evaluation of 15N abundance of N derived from biological N2 fixation (BNF) in legume shoots (‘BS’ value) is essential for quantifying BNF inputs to field-grown legumes. The aim of this study was to investigate the impact of shading, development stage of soybean (Glycine max) and rhizobium strain on the ‘BS’ value.Methods: Soybean plants were grown in pots of autoclaved sand/perlite mixture in the open field. Plants were harvested at weekly intervals from 46 days after planting (DAP) to 75 DAP. All material was analysed for N and 15N abundance. ‘BS’ was calculated assuming 50% of seed N was translocated to the shoots.Results: ‘BS’ was stable until 60 DAP but subsequently increased for the three strains tested. Nodule efficiency (N2 fixed g DM nodule−1) was greatly increased by shading and was significantly different between Bradyrhizobium species. ‘BS’ was greatly increased by shading.Conclusion: We recommend that ‘BS’ should be evaluated on plants of the same development stage and light intensity as those where BNF is quantified in the field. Different Bradyrhizobium strains make a large impact on ‘BS’ and may lead to considerable errors in estimation of BNF inputs to plants with high %N derived from BNF.