PFKFB4 and cancer: Using multiple cell types and approaches, we found that: (i) recombinant human PFKFB4 has far more kinase activity than phosphatase activity (kinase:phosphatase ratio = 4.3:1); (ii) selective inhibition of PFKFB4 using two unique siRNA molecules reduces the intracellular F2,6BP in six of seven cancer cell lines indicating that endogenous PFKFB4 synthesizes F2,6BP in these cancer cells; (iii) over-expression of PFKFB4 increases the F2,6BP in all examined cancer cell lines; and (iv) genomic deletion of the Pfkfb4 gene reduces F2,6BP in LT-immortalized fibroblasts.