The synthesis of glycosylated haemoglobins in vivo was measured during 24 h of controlled hyperglycaemia in seven insulin dependent diabetics. The mean blood glucose concentration was 22 mmol/l, while electrolytes and other metabolites were kept normal by infusion of 4-23 IU of insulin during hyperglycaemia. The study confirmed the velocity and magnitude of unstable HbAlc formation previously found in vitro. The stable HbAlc formed in 24 h was an average 0.006% of total haemoglobin/mmol glucose. This compares well with the rate of HbAlc synthesis reported in normal subjects using 59Fe-kinetic measurements, and is in accordance with the concept of slow changes in stable HbAlc with time and glucose concentration. To investigate the possibility that the rate of HbAlc synthesis varies with erythrocyte age, glycosylated haemoglobins were measured in erythrocyte fractions after density separation on Percoll-Albumin gradients. We found both in normal subjects and in insulin treated diabetics that the 5% least dense cells contained 70%-80% of whole blood HbAlc. Assuming the least dense cells to be the youngest erythrocytes, this observation is inconsistent with a slow linear increase in HbAlc. Similar results were obtained in six newly diagnosed insulin dependent diabetic patients both before and after the first 30 days of insulin treatment, even though a marked decrease in young cell HbAlc would be expected with the improved glucose control observed. We therefore conclude that density separation of erythrocytes is an inadequate technique to study age related HbAlc synthesis.