To determine red cell age-related changes in the formation of glycosylated (glyco) hemoglobins (Hbs), red cells were fractionated by dextran 40 density gradient centrifugation and glyco Hbs determined by both Bio-Rex 70 cation exchange and phenylboronate-agarose affinity chromatographic methods. Blood samples from a normal adult, adults with White's class A diabetes and insulin dependent diabetes mellitus (IDDM), and newborns of normal and diabetic women were used for this study. Red cell fractions from the newborns and the adults showed a steady increase in the levels of glyco Hb determined by the affinity method with increasing red cell density. In the newborns, the absolute levels and the increase of glyco Hb were smaller than in the adults, probably because of decreased red cell survival in fetuses compared to adults. The glyco Hb value for each cell fraction was significantly higher in the adult with IDDM and in the newborns of diabetic women. In the adults, the levels of Hb Ala + b and Hb Alc determined by Bio-Rex 70 chromatography also showed an increase with increasing red cell age. In the newborns, Hb Flc, which contains predominantly acetylated and some glycosylated Hb F, showed only a small increase with increasing red cell age over and above that due to a concomitant increase in Hb F. On the contrary, Hb Fla + b, which contains mostly glyco Hb F, showed a larger increase. The results confirm that glycosylation, but not acetylation, of Hb F takes place over the entire lifespan of red cells.