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• Long-term soybean breeding for yield has increased plant N demands. • Total shoot N and N from BNF increased with cultivar year of release. • Sensitivity of BNF to soil mineral N has not changed over years of soybean breeding. Long-term soybean [ Glycine max (L) Merr.] breeding for yield has increased plant nitrogen (N) demands. On one hand, because N fertilizer application in soybean production systems continues to be insignificant, increased plant N demands over time may have been satisfied from greater biological N fixation (BNF). On the other hand, increased soil residual N over time may have affected the sensitivity of nodulation and BNF. To understand the impact of breeding for greater yield and the effect of soil residual N on nodulation and BNF, two field and a greenhouse study were conducted. Field studies were conducted with maturity group IV soybean cultivars released from 1930 to 2005 and included experiments in four environments. Total shoot N and N from BNF increased with cultivar year of release in two of the four environments. Simulation of different levels of residual soil mineral N by application of 0, 34, 67, and 101 kg N ha−1 shortly after planting resulted in linear increases in shoot N content and δ15N, and linear decreases in nodule number and nodule dry matter in the field. Consistent with these results, fertigation of greenhouse-grown soybean cultivars with different levels of NH 4 NO 3 led to a reduction in nodule number, dry matter, and size. Overall, results from these studies indicate that increases in seed yields with cultivar year of release were associated with greater amounts of N derived from BNF as well as greater total shoot N accumulation, but the relative contribution of BNF to total shoot N did not change over time. Analysis also suggest that the sensitivity of nodulation and BNF to soil mineral N has not been altered over the course of soybean breeding. [ABSTRACT FROM AUTHOR]