Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and 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. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in which, at most, only a single domain would be active. Two of the T. brucei proteins showed most conservation in the PFK-2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase-2 domain. The two PFK-2-like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme purified from T. brucei, whereas no activity was found for the second. Interestingly, several of the predicted trypanosomatid PFK-2/FBPase-2 proteins have long N-terminal extensions. The N-terminal domains of the two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are known to mediate protein-protein interactions. Phylogenetic analysis suggests that the four different PFK-2/FBPase-2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage. A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin-motif repeats in the PFK-2 isoenzymes is presented.