Mature erythrocytes, when removed from the circulation, exhibit severe disturbances of glycolytic flow, with accumulation not only of lactate, the ultimate product of glycolysis, but also of several upstream metabolic intermediates, primarily fructose-1,6-diphosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate. This accumulation may be prevented (and also reverted) by allowing the diffusible end products lactate and pyruvate to leave the cell by equilibrating with a much larger extracellular compartment. The disturbance of erythrocyte glycolysis does not result from direct inhibition by lactate itself but from the interplay between the lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (3-PGAD) reactions. The accumulation of intermediates reflects the increased lactate-to-pyruvate ratio; this leads to a secondary imbalance of the nicotinamide adenine dinucleotide-to-reduced nicotinamide adenine dinucleotide (NAD-to-NADH) ratio, which in turn slows down glycolysis at the 3-PGAD step, whose upstream metabolites then pile up. No accumulation, however, takes place if the lactate-to-pyruvate ratio is maintained constant in the extracellular compartment, regardless of concentrations. These studies demonstrate that orderly glycolysis in the erythrocyte is regulated by the NAD-to-NADH ratio and also provide a method that makes possible the in vitro study of erythrocyte glycolysis.