AbstractHuman erythrocytes were separated by buoyant density ultracentrifugation into fractions of progressively increasing mean cell age to measure the changes in glycolytic activity that occur during their 120‐day life‐span. The maximal activities of all glycolytic enzymes were shown to decline exponentially with cell age. Only three glycolytic enzymes exhibited a marked rate of decline with a t1/2shorter than the cell life‐span: hexokinase, aldolase, and pyruvate kinase.Glucose utilization, when measured in steady‐state conditions (1 mM inorganic phosphate), showed a fourfold decrease through the erythrocyte life‐span; lactate production also declined, but at a slower rate. When incubating conditions were altered by the introduction of a metabolic stimulus (either high phosphate for glycolysis, or methylene blue for the pentose pathway) the youngest cell fractions responded with decidedly increased rates of glucose consumption and lactate production. However, this ability gradually declined with cell aging, and ultimately, the oldest cells had metabolic rates as low as if there were no stimulus present. The oldest erythrocytes appear to have lost the flexibility needed to respond to metabolic stress and are more vulnerable to events in the circulation that may require the ability to increase the basal rate. This defect is probably responsible for the disappearance of aged erythrocytes from the ci