A B S T R A C T Human phosphofructokinase (PFK; EC 2.7.1.11) exists in tetrameric isozymic forms. Muscle and liver contain the homotetramers M4 and L4, whereas erythrocytes contain five isozymes composed of M (muscle) and L (liver) subunits, i.e., M4, M3L, M2L2, ML3, and L4. Inherited defects of erythrocyte PFK are usually partial and are described in association with heterogeneous clinical syndromes. To define the molecular basis and pathogenesis of this enzymopathy, we investigated four unrelated individuals manifesting myopathy and hemolysis (glycogenosis type VII), isolated hemolysis, or no symptoms at all.The three symptomatic patients showed high-normal hemoglobin levels, despite hemolysis and early-onset hyperuricemia. They showed total lack of muscle-type PFK and suffered from exertional myopathy of varying severity. In the erythrocytes, a metabolic crossover was evident at the PFK step: the levels of hexose monophosphates were elevated and those of 2,3-diphosphoglycerate (2,3-DPG) were depressed, causing strikingly increased hemoglobin-oxygen affinity. In all cases,