Measurement of intracellular metabolites and glycolytic activities in enzyme-deficient red cells has proved to be a valuable approach to studying the mechanism of hemolysis and metabolic regulations. Glycolytic intermediates, nucleotides, glucose consumption, and lactate formation were measured in phosphohexose isomerase, phosphoglycerate kinase, diphosphoglycerate mutase, and pyruvate kinase deficiencies. Pyruvate kinase deficiency was the only defect which was found to impair glycolytic activity, since there is no metabolic bypass at this level. Pyruvate kinase and phosphoglycerate kinase deficiencies may produce hemolysis through a decreased ADP phosphorylation to ATP. Defects of phosphoglycerate kinase and diphosphoglycerate mutase modify 2,3-diphosphoglycerate levels, but self-regulating mechanism limit these variations which alter the oxygen affinity of hemoglobin. The mechanism of hemolysis has not been explained definitely in diphosphoglycerate mutase and phosphohexose isomerase deficiencies; however, in the latter case it has been tentatively postulated that the compensatory bypass through the pentose-phosphate pathway may decrease progressively in aging cells and thus shorten their lifespan.