The large nitrogen (N) demand of high yielding soybeans is met by soil and symbiotically fixed N, but the relative contribution of fixed N at high yield levels is not clear. The objective of our work was to evaluate whether the application of fertilizer N during the crop cycle increases total N uptake and to what extent N fixation can be complemented with fertilizer N. Four N fertilizer treatments were superimposed on two contrasting nutrient management backgrounds (M1=recommended nutrient management; M2=intensive nutrient management); N treatments were: No N fertilization (N1), 180 kg ha-1 as polymer-coated urea placed 20 cm below the surface before planting (N2); 180 kg N ha-1 as nitrate ammonium on the surface before planting and at V6 (N3) and 180 kg N ha-1 as nitrate ammonium on the surface at R5 (N4). Nitrogen fixation was determined by the relative ureides content in sap. Grain yield in N1 was 4995 kg ha-1, and a 5% increase in grain yield was observed with the late N supply strategies (N2+N4). This response was accompanied by an 8% increase in total N uptake. N fixation was negatively affected by N application on the surface. A negative exponential relationship was found between nitrate content in the upper 5 cm of soil and N fixation. This pool accounted for 42% of total N uptake in N1; however it was only 25% in N3 and N4. On the other hand, N2 had higher N uptake than N1 and maintained a high proportion of fixed N. Intensive management background with higher soil-NO3 content at planting suppressed N fixation by 26%. These results showed a slight increase in grain yield for grain yields above 5000 kg ha-1and the possibility of counteracting nitrate suppression of N fixation by placing slow-release N fertilizer below the main nodulation area.