African trypanosome species are causative agents for sleeping sickness in humans and nagana disease in cattle. Trypanosoma brucei can generate ATP via a reverse reaction with glycerol kinase (GK) when alternative oxidase (AOX) is inhibited; thus, GK is considered to be a crucial target for chemotherapy combined with AOX. However, the energy metabolism systems of African trypanosome species other than T. brucei are poorly understood. Thus, GK genes were surveyed from genome databases and cloned by PCR from T. vivax and T. congolense. Then, recombinant GK proteins (rGK) of T. vivax, T. congolense and T. brucei were expressed and purified. Kinetic analysis of these rGK proteins revealed that the K(m) values of T. congolense rGK for ADP and G-3-P substrates were lower than those of T. vivax and T. brucei. The expression level of GK molecules was highest in T. congolense cells and lowest in T. vivax cells. Based on these results, effective combination dosages of ascofuranone, a specific inhibitor of AOX, and glycerol, an inhibitor of the GK reverse reaction, were determined by using in vitro-cultured trypanosome cells.