The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.