The pKa values have been determined for eight of the nine histidine residues and the amino terminus of the N-lobe of human apo-transferrin (hTF/2N), and for seven of the nine histidine residues and the amino terminus of the protein Asp63Ser hTF/2N containing a mutation of the Fe3+-ligand Asp63 to Ser63. Calculations suggested that substitution of aspartate by serine would result in decreases of the pKa values of most of the histidine residues in the protein. This was found to be the case experimentally, and allowed assignment of the εCH resonance of His249. For the wild-type protein, the His residue with a pKa of 7.40 was assigned as His249, whereas for the mutant, no observable His residue had a pKa value higher than 6.9. The protonated form of His249 appears to be stabilised by interactions with Asp63, and the high pKa value may be critical for ensuring the release of iron at endosomal pH (5.5). The mutation lowered the apparent binding constant of hTF/2N for the synergistic anion oxalate from logK 4.0 to logK 3.3. 1H NMR spectral changes induced by Ga3+ binding to the mutant are compared to those observed for the wild-type protein.