6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is a bifunctional enzyme, which is responsible for maintaining the cellular level of fructose-2,6-bisphosphate, a powerful allosteric activator of glycolysis. We describe herein the overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 (PFKFB-4) isozyme in the human breast and colon malignant tumors as compared to corresponding non-malignant tissue counterparts. We have shown also that breast malignant cell line MCF7 constitutively express PFKFB-4 mRNA and that the expression of this gene is highly induced by hypoxia. Overexpression of PFKFB-4 transcript levels in breast and colon malignant tumors correlates with enhanced expression of PFKFB-3, hypoxia-inducible factor (HIF)-1alpha and known HIF-1 dependent genes glucose transporter 1 (Glut1) and vascular endothelial growth factor (VEGF). Thus, our data clearly demonstrates overexpression of PFKFB-4 mRNA and protein in the breast and colon malignant tumors.