||Trypanosoma brucei , the single-celled eukaryotic parasite responsible for African sleeping sickness, has an unusual β-hydroxybutyrate dehydrogenase. His-tagged recombinant protein was expressed in E. coli and purified to approximate homogeneity. Kinetic studies were then carried out to determine the kinetic parameters for substrates and inhibitors. The K M and specific activity for Tb βHBDH with acetoacetate as a substrate were determined to be 0.69±0.16 mM and 0.38±0.058 ìmol/min*mg, respectively. With D-3-hydroxybutyrate as a substrate, the K M and specific activity for TbβHBDH were determined to be 0.65±0.34 mM and 22.25±2.25 μmol/min*mg. TbβHBDH utilizes both NADH and NADPH equally well as cofactors, and the enzyme exhibits cooperativity when binding to NAD + and NADP + . Both properties are distinct to the trypanosome enzyme. Size exclusion chromatography suggests that the oligomeric state of the enzyme was tetramer. Finally, RNA interference studies, verified by Western blot analysis, indicated an important physiological role of Tb βHBDH in the parasites.