Nitrogen metabolism differences, including N2 fixation, between high‐and normal‐seed‐protein soybean [Glycine max (L.) Merr.] lines of equivalent plant maturity and seed yielding ability have not been determined in field tests. A replicated field experiment was conducted in 1985 and 1986 to determine such differences on a Mattapex silt loam (fine, silty, mixed, mesic Aqualfic Hapludult) with low soil N. The study used the nodulating‐nonnodulating N difference method to estimate N2 fixation of two soybean lines with contrasting seed protein levels. Eight sequential harvests were conducted from R5 (beginning seed) to combine harvest for the dry weights and N contents of vegetative and reproductive portions of the plants. There was no significant difference between the 2‐yr mean seed yields of the high‐ and normal‐protein lines, but the high‐protein line was 2 d later in maturity. The high‐protein line accumulated more N, fixed more N2, and remobilized more N to the seed than did the normal‐protein line (231 vs. 194, 114 vs. 77, and 87 vs. 76 kg ha−1, respectively). The greater N accumulation by the high‐protein line resulted from N2 fixation being sustained until the late R6 (full seed) stage. Nodule occupancy by strains of Bradyrhizobium japonicum was similar for the high‐ and normal‐seed‐protein lines. Thus, the ability to sustain N2 fixation until later stages of reproduction is a host plant attribute that may contribute to the greater N metabolism of a high‐seed‐protein soybean.