The phosphofructokinase C isozyme (PFK-C) from ascites tumor cells has been cloned and characterized to investigate the particular properties of PFK activity in this type of cells. The isolated cDNA encodes a protein of 784 amino acids and 85.5 kDa, whose expression was constant along tumor growth and markedly decreased when cell proliferation stops. The enzyme was functionally expressed in a PFK-deficient strain of Saccharomyces cerevisiae and purified to homogeneity, Recombinant PFK-C exhibited the same subunit size as the tumor wild-type isozyme and its steady-state kinetic parameters were similar to those of the form present in normal cells. The regulatory properties of the C isozyme accounted for the lack of fructose-1,6-P-2 activation and the P-enolpyruvate inhibition of PFK activity observed in ascites tumor preparations containing the various isozyme types. Nevertheless, PFK-C binds fructose-1,6-P-2 to an allosteric site as suggested by protection against thermal denaturation. Our results indicate that glucose metabolism in tumor cells is not regulated by a mutant form of PFK-C but by a high level expression of the normal C isozyme,