||6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional tissue-specific enzyme involved in the regulation of glycolysis and gluconeogenesis. Evolutionarily, mammals acquired four different isoforms of the PFK-2/FBPase-2 enzyme corresponding to four mammalian tissues: skeletal muscle, heart, liver and brain. Interestingly, the unicellular Trypansoma brucei parasite, the causative agent of African sleeping sickness, also has four PFK-2/FBPase-2 paralogs. However, two of these paralogs appear to be catalytically inactive. The remaining two trypanosome paralogs appear to be monofunctional, possessing either kinase (PFK-2) or bisphosphatase (FBPase-2) activity. In the work described herein, all four paralogs were analyzed in vivo by RNA interference (RNAi) studies. Four separate trypanosome knock-down cell lines, corresponding to each paralog, were generated. Growth studies of these parasites were carried out in rich media (SDM-79), minimal media (ME-83) and minimal media lacking glucose (ME-83 - glucose). Under these various conditions, altered growth was noted for each cell line, including the catalytically inactive paralogs.