Summary The contribution of large molecular size organic nitrogen (N) to plant N uptake is unclear. Soils with and without maize, at three pH levels, were treated with (carbon‐14 and ‐13 (14C, 13C), 15N) triple‐labelled > 100 kDa organic N. After 48 h, soil and maize were sampled for bulk and compound specific isotope analysis to study the turnover in soil and plant 13C and 15N uptake. Mineralization of > 100 kDa organic N increased with higher pH only in soil without maize. The > 100 kDa organic N disappeared rapidly in soils with and without maize, but surprisingly more > 100 kDa organic N derived amino acids remained in soil with than without maize – most likely in the microbial biomass. Total 15N uptake in maize increased with higher soil pH. The organic N uptake was estimated to account for 20–30% of the total 15N uptake. Organic N uptake was confirmed by the presence of 13C‐labelled amino acids in maize roots. The study suggests that the importance of plant organic N uptake increases when N is derived from complex molecules such as proteins compared to studies using single amino acids as N source, and that rhizosphere microorganisms increase anabolic utilization of organic N compared to microorganisms in the bulk soil.