In vivo drug susceptibility tests involving treatment of infected mice and cattle were performed on two trypanosome stocks, a T. brucei brucei and a T.b. rhodesiense, isolated in South Eastern Uganda. The T. b. rhodesiense stock had expressed reduced susceptibility to diminazene aceturate and isometamidium chloride in vitro, while the other, a T. b. brucei stock was susceptible. Diminazene aceturate at 14 mg/kg was not sufficient to cure all T. b. rhodesiense infected mice. Similarly, in the case of isometamidium chloride, 33% of infected mice treated with 2.0 mg/kg drug were not cured. In contrast, mice infected with the susceptible T. b. brucei and treated similarly with either drug were all cured. However, when cattle infected with the T. b. rhodesiense stock, or the susceptible T. b. brucei stock, or a 1:1 mixture of the two were treated with 7 mg/kg diminazene aceturate, they were all cured. Use of diagnostic PCR employing T. brucei specific primers confirmed that although the cattle had acquired infection pre-treatment, no trypanosome DNA amplification signal was demonstrated in the samples collected 60 days post-treatment. The reduced susceptibility of this T. b. rhodesiense, which could be demonstrated in mice as well as in culture, may indicate the existing potential for evolution of resistance in South Eastern Uganda.