The existence of a five-membered isozyme system for human phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been demonstrated. These multimolecular forms result from the random polymerization of two distinct subunits, M (muscle type) and L (liver type), to form all possible tetrameters-i.e., M(4), M(3)L, M(2)L(2), ML(3), and L(4). Partially purified muscle and liver PFKs were hybridized by dissociation at low pH and then recombination at neutrality. Three hybrid species were generated in addition to the two parental isozymes, to yield an entire five-membered set. The various species could be consistently and reproducibly separated from one another by DEAE-Sephadex chromatography at pH 8.0 with a concave elution gradient of salt. Under similar experimental conditions, erythrocyte PFK from hemolysates was also resolved into five species chromatographically indistinguishable from those produced in the above experiment. Immunological and kinetic studies of the isozymes provided corroborative evidence to support the proposed subunit structures. Erythrocyte PFK was found to have kinetic properties intermediate between those of muscle and liver PFK and was neutralized only 50% by an antiserum against muscle PFK that completely neutralized muscle PFK. These data demonstrate that muscle and liver PFKs are distinct homotetramers-i.e., M(4) and L(4), respectively-whereas erythrocyte PFK is a heterogeneous mixture of all five isozymes. The structural heterogeneity of erythrocyte PFK provides a molecular genetic basis for the differential organ involvement observed in some inherited PFK deficiency states in which myopathy or hemolysis or both can occur.