Citation: Anglade, J., G. Billen, and J. Garnier. 2015. Relationships for estimating N 2 fixation in legumes: incidence for N balance of legume-based cropping systems in Europe. Ecosphere 6(3):37. http://dx.doi.org/10.1890/ES14-00353.1Abstract. Management of nitrogen (N) inputs into agricultural systems is of increasing interest due to the intensive use of synthetic fertilizers in modern cash crop systems, which has caused considerable environmental damage, including nitrate contamination of surface and groundwater, eutrophication of coastal marine areas and emission of greenhouse gases. The use of legumes grown in rotations or intercropping is nowadays regarded as an alternative and sustainable way of introducing N into low-input cropping systems. In order to develop a simple tool to assess biological nitrogen fixation (BNF) by legume pulse and forages within crop rotations, we conducted a systematic literature survey with a quantitative statistical analysis intended to define a robust relationship between measurable variables and N 2 fixation. We found highly significant correlations between total N accumulation in shoot matter and fixed N 2 in forage (y ¼ 0.79x À 0.49) and grain (y ¼ 0.70x þ 1.01) legumes. Accurately estimating the contribution of belowground components is more difficult because of a lack of relevant studies. Properly assessing BNF is crucial to evaluate the possibility of diversified legume based cropping systems achieving a balance between inputs and outputs, thus ensuring both short-term productivity and long-term sustainability. For a typical nine-year organic rotation in the Paris basin alternating legumes and cereals, the soil surface balance, calculated with the relationships obtained, indicated that N fluxes were balanced, leaving a surplus of only a few kg N/ha/yr, with legumes accounting for 86% of total N inputs.