Red blood cell (RBC) fractions were studied after separation of whole blood by means of counterflow centrifugation, Percoll column (Pharmacia, Uppsala, Sweden), and a combination of both separation techniques. Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hemoglobin A1c (HbA1c) were measured in each fraction. From the results it was obvious that the combination of both techniques was the best separation technique of these three. MCV had a good correlation with cell age as measured with HbA1c concentration gradient; MCH and MCHC less so. MCV and MCH decreased in parallel to an increase in HbA1c. MCHC increased with increasing HbA1c. From these data it is concluded that there is a steadily ongoing loss of cellular hemoglobin and proportionally more cellular water during the life of the RBC.