A variant hemoglobin was found in a Japanese female whose hemoglobin was studied to clarify the cause of a low Hb A1c value, found during a routine medical examination. The detection and identification of the variant was performed by electrospray ionization mass spectrometry. Its structure was revealed to be the same as Hb Peterborough [beta 111(G13)Val-->Phe]. For sequence determination, oxidized globin as well as non-derivatized globin were cleaved by trypsin and lysyl endopeptidase. An abnormal peptide was found in digests of oxidized globin, as shown by electrospray ionization mass spectrometry. Cysteic acid in oxidized peptides enhanced the abundance of fragment ions in tandem mass spectrometry, which helped to quickly and accurately determine the substitution in beta T-12, a peptide in the core region. Electrospray ionization mass spectrometry analysis of the hemolysate also showed a low level of glycated hemoglobin. The patient's hemolysate showed decreased stability in the isopropanol test. An abnormal band was detected on isoelectrofocusing on the cathodic side of normal Hb A. This is the second report of Hb Peterborough and the first of its occurrence in Japan.