Animal trypanosomiasis is a major hinderance to the growth of livestock farming in sub-Saharan Africa. Chemotherapy using isometamidium, diminazene and ethidium bromide has been the main control method in the absence of a vaccine against this disease. The effectiveness of these few trypanocides is severely threatened by the widespread development of resistance. Therefore, an understanding of the mechanism(s) involved in the development of resistance will assist in the development of screening protocols for easy identification of resistant cases prior to treatment, and also in finding ways to reverse the resistance. We studied the mechanism of resistance to isometamidium in bloodstream forms of Trypanosoma brucei. Resistance to isometamidium in Trypanosoma brucei was found to be composed of a reduced uptake of the drug and the modification of the F1F0 ATPase complex; active drug efflux by ABC transporters was not involved in the resistance mechanism, although efflux of ISM could be observed in both wild-type and resistant lines. Expression of the transporter gene TbAT1, as well as of TbAT-E and TbAT-A, in yeast, each resulted in increased ISM uptake. In addition, the Vmax for the LAPT1 drug transport activity (Low Affinity Pentamidine Transporter) in ISM-resistant trypanosomes (clone ISMR1) was significantly reduced (P<0.05; Student’s t-test) compared to the wild type control. Also, two point mutations, namely G37A and C851A were found in the ATP synthase gamma subunit of the F1F0 ATPase complex of isometamidium-resistant trypanosomes. The resistant clones also lost their mitochondrial DNA and mitochondrial membrane potential and displayed various levels of cross-resistance to ethidium, diminazene, pentamidine and oligomycin. The C851A mutation introduced a stop codon in the open reading frame of the ATP synthase gamma gene. This mutation, when introduced into the wild type Tb427, produced resistance to isometamidium, and cross resistance to diminazene, ethidium, pentamidine and oligomycin. C851A-ATP synthase gamma proves to be a dominant mutation that allows the rapid loss of mitochondrial DNA after just three days exposure of the parasites to 20 nM ISM or ethidium bromide. Finally, following a recent genome-wide loss-of-function RNAi screen that linked TbAQP2 with pentamidine and melarsoprol cross resistance, we were able to demonstrate that TbAQP2 encodes the HAPT1 in T. brucei, thus leaving us with the LAPT1 as the only known T. b. brucei drug transporter of unknown genetic origin. We however identified specific inhibitors for this transporter (LAPT1) that will be of use in its further characterization.