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Analyses were made on the adenosine transporter-1 gene in Trypanosoma brucei (TbAT1), encoding a P2-like nucleoside transporter, from T. brucei brucei field stocks to investigate a possible link between the presence of mutations in this gene and isometamidium resistance. We have analysed the gene from 11 isometamidium-sensitive field stocks isolated from cattle in Uganda, two sensitive reference clones and two resistant reference clones. Sequence alignment showed that the isometamidium-sensitive T. b. brucei contained the wild-type sequence patterns. In contrast, the isometamidium-resistant T. b. brucei stocks showed the mutant-type sequence patterns with six point mutations that had previously been reported in a laboratory-derived arsenical-resistant T. brucei strain. In order to analyse the RFLP pattern of a fragment of TbAT1 (nucleotides 430-1108), the 677 bp PCR products from eight of the isometamidium-sensitive and two of the isometamidium-resistant T. b. brucei were subjected to digestion with Sfa NI. The results revealed two different banding patterns: the digest resulted in fragment sizes of 566 and 111 bp in the case of TbAT1 from isometamidium-sensitive stocks, whereas it produced fragment sizes of 435 and 242 bp in the case of TbAT1 from isometamidium-resistant stocks. Thus, the isometamidium-sensitive and resistant T. b. brucei could be successfully distinguished by digestion with the restriction endonuclease Sfa NI.