Maximising NO3 - uptake during seedling development is important as it has a major influence on plant growth and yield. However, little is known about the processes leading to, and involved in, the initiation of root NO3 - uptake capacity in developing seedlings. This study examines the physiological processes involved in root NO3 - uptake and metabolism, to gain an understanding of how the NO3 - uptake system responds to meet demand as maize seedlings transition from seed N use to external N capture. The concentrations of seed-derived free amino acids within root and shoot tissues are initially high, but decrease rapidly until stabilising eight days after imbibition (DAI). Similarly, shoot N% decreases, but does not stabilise until 12-13 DAI. Following the decrease in free amino acid concentrations, root NO3 - uptake capacity increases until shoot N% stabilises. The increase in root NO3 uptake capacity corresponds with a rapid rise in transcript levels of putative NO3 - transporters, ZmNRT2.1 and ZmNRT2.2. The processes underlying the increase in root NO3 - uptake capacity to meet N demand provide an insight into the processes controlling N uptake.