Increasing nitrogen supply can increase Fe and Zn concentrations in wheat grain, but the underlying mechanisms remain unclear. Size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry was used to determine Fe and Zn speciation in the soluble extracts of grain pearling fractions of two wheat cultivars grown at two N rates (100 and 350 kg of N ha-1). Increasing N supply increased the concentrations of total Fe and Zn and the portions of Fe and Zn unextractable with a Tris-HCl buffer and decreased the concentrations of Tris-HCl-extractable (soluble) Fe and Zn. Within the soluble fraction, Fe and Zn bound to low molecular weight compounds, likely to be Fe-nicotianamine and Fe-deoxymugineic acid or Zn-nicotianamine, were decreased by 5-12% and 4-37%, respectively, by the high N treatment, whereas Fe and Zn bound to soluble high molecular weight or soluble phytate fractions were less affected. The positive effect of N on grain Fe and Zn concentrations was attributed to an increased sink in the grain, probably in the form of water-insoluble proteins.