We have attempted to separate various hemoglobins in 10 normal and 11 diabetic persons by Bio-Rex 70 chromatography at 4°C with an exponentially increasing sodium phosphate gradient. Minor hemoglobins, namely A Ia1, A Ia2, A Ib1, A Ib2, A Ib3, A Ic, A Id1, A Id2 and A Id3, have been separated and eluted in that order. Most of these minor hemoglobins were newly observed and could not be detected by the previous chromatographic techniques. On the basis of colorimetric assay protein-bound ketoamine was present in all the minor hemoglobins, which was confirmed by chromatographic separation of hemoglobins after reduction with NaB 3H 4. All the minor hemoglobins, with the exception of Hb A Ia1, Hb Ia2, and Hb A Ib1, showed a 2-fold increase in the diabetic patients. Hb A Ic (5.0% in normals; 9.0% in diabetics) and Hb A Id3 (1.9% in normals; 3.2% in diabetics) were present in the largest amounts. Both Hb A Id3 and Hb A o showed a decrease in oxygen affinity in the presence of 2,3-diphosphoglycerate, whereas Hb A Ic showed no effect. Separation of globin chains by cellulose acetate electrophoresis at pH 8.6 showed that the cathodal mobility of the α chains of Hb A Id3 was slower than those of Hb A 0. Glycosylation of Hb with [ 14C]glycose followed by separation of hemoglobins by two Bio-Rex 70 chromatographic methods indicated that the minor Hb formed by glycosylation of the α-chain amino-terminus was separated from Hb A 0, while the minor hemoglobins formed by the glycosylation of ϵ-NH 2 groups chromatographed with Hb a 0.