Hb Auckland is a newly described unstable hemoglobin with a mutation of alpha 97(F8)His-->Asn. This substitution, involving the proximal histidine, does not lead to methemoglobinemia, but to instability and accelerated heme loss. The clinical picture is of a mild compensated hemolytic anemia. The presence of an abnormal hemoglobin was first demonstrated by the isopropanol stability test and confirmed by electrospray ionization mass spectrometry of total lysate. This showed that 14% of the alpha chains had a mass of 15,103.4 Da, i.e. 23 Da less than normal. Examination of tryptic digests showed an identical decrease in mass for peptide alpha T-9 (from 2,997.4 to 2,974.5 Da). Subdigestion with endoproteinase Asp-N located the 23 Da loss to residues alpha 85-90, and further digestion with thermolysin identified the mutation as His-->Asn at position 87 of the alpha chain. This was confirmed by sequence analysis of the peptide alpha 85-90.