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African animal trypanosomes cause a fatal disease commonly called nagana in domestic livestock. Trypanosoma congolense, T. vivax and T. brucei are the main trypanosome species responsible for the disease in livestock. The disease is considered a major constraint to increasing livestock production in sub-Saharan Africa. Isometamidium is the only recommended prophylactic drug and is widely used in the treatment of trypanosome infections in cattle and small ruminants. Resistance to isometamidium by T. brucei has been reported from different parts of Africa. The in vivo and in vitro techniques commonly used to identify drug resistance in trypanosomes suffer from a number of drawbacks. Simpler, more sensitive, fast and reliable methods for typing drug-resistant trypanosomes are required. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. Other workers have previously cloned the adenosine transporter-1 gene, TbAT1, from T. b. brucei, and they implicated mutation of this gene for resistance to arsenicals. In the current study, attempts are made to investigate possible links between mutations in TbAT1 gene and isometamidium resistance in T. b. brucei. We have analysed the TbAT1 gene of T. b. brucei from 11 isometamidium sensitive field stocks, 2 sensitive reference clones and 2 resistant reference clones. All of the field stocks and both of the sensitive reference clones had similar sequence patterns, which corresponded to the melasoprol-sensitive wild-type T. brucei. In contrast, isometamidium resistant clones had a mutated TbAT1. Both clones had the same set of point mutations described earlier in melasoprol-resistant T. b. rhodesiense and T. b. gambiense.