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Although relationships among soybean (Glycine max [L.] Merr) seed yield, nitrogen (N) uptake, biological N2 fixation (BNF), and response to N fertilization have received considerable coverage in the scientific literature, a comprehensive summary and interpretation of these interactions with specific emphasis on high yield environments is lacking. Six hundred and thirty-seven data sets (site–year–treatment combinations) were analyzed from field studies that had examined these variables and had been published in refereed journals from 1966 to 2006. A mean linear increase of 0.013 Mg soybean seed yield per kg increase in N accumulation in above-ground biomass was evident in these data. The lower (maximum N accumulation) and upper (maximum N dilution) boundaries for this relationship had slopes of 0.0064 and 0.0188 Mg grain kg−1 N, respectively. On an average, 50–60% of soybean N demand was met by biological N2 fixation. In most situations the amount of N fixed was not sufficient to replace N export from the field in harvested seed. The partial N balance (fixed N in above-ground biomass − N in seeds) was negative in 80% of all data sets, with a mean net soil N mining of −40 kg N ha−1. However, when an average estimated below-ground N contribution of 24% of total plant N was included, the average N balance was close to neutral (−4 kg N ha−1). The gap between crop N uptake and N supplied by BNF tended to increase at higher seed yields for which the associated crop N demand is higher. Soybean yield was more likely to respond to N fertilization in high-yield (>4.5 Mg ha-1) environments. A negative exponential relationship was observed between N fertilizer rate and N2 fixation when N was applied on the surface or incorporated in the topmost soil layers. Deep placement of slow-release fertilizer below the nodulation zone, or late N applications during reproductive stages, may be promising alternatives for achieving a yield response to N fertilization in high-yielding environments. The results from many N fertilization studies are often confounded by insufficiently optimized BNF or other management factors that may have precluded achieving BNF-mediated yields near the yield potential ceiling. More studies will be needed to fully understand the extent to which the N requirements of soybean grown at potential yields levels can be met by optimizing BNF alone as opposed to supplementing BNF with applied N. Such optimization will require evaluating new inoculant technologies, greater temporal precision in crop and soil management, and most importantly, detailed measurements of the contributions of soil N, BNF, and the efficiency of fertilizer N uptake throughout the crop cycle. Such information is required to develop more reliable guidelines for managing both BNF and fertilizer N in high-yielding environments, and also to improve soybean simulation models.