Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy meta-bolism in these protozoan parasites. A 6-phosphofructo-2-kinase, respon-sible for the synthesis of this regulator, was highly purified from the bloodstream form ofTrypanosoma bruceiand kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase⁄fructose-2,6-bisphosphatase (PFK-2⁄FBPase-2) were found for both T. .