Breeding for high seed protein concentration in soybean [Glycine max (L.) Merrill] often results in lower yield, but the basis for this negative relationship is not well understood. To address this question, we evaluated the N acquisition characteristics of three high protein and three normal soybean genotypes in the field for 3 years. Plants were grown in 0.76 m rows following conventional cultural practices and water stress was minimized with sprinkler irrigation. We determined the mass and N concentration of leaves, petioles and stems at the beginning of seed filling (growth stage R5) and of stems at maturity. The N concentration of abscised leaves and petioles was also determined. There was significant variation among genotypes in total seed N (g m−2) at maturity (range from 14.7 to 24.4 g N m−2) as a result of variation in seed N concentration and yield. There was no evidence that the larger amounts of mature seed N were associated with a larger vegetative N reservoir at growth stage R5 as determined by vegetative mass at R5 or the concentration of N in vegetative tissues. Increasing seed N at maturity did not lower the N concentration in abscised leaves and petioles, or in the stems at maturity. The rate and timing of leaf senescence (loss of chlorophyll) was essentially the same for all genotypes. With no increase in the contribution from redistributed N, increases in N uptake or fixation during seed filling must have been responsible for the higher levels of seed N at maturity in high-protein genotypes. These data suggest that increasing total seed N at maturity by selecting for higher seed protein concentration or higher yield in soybean does not require, as some models suggest, a larger vegetative N reservoir at the beginning of seed filling or more rapid senescence.